Oligooxopiperazines and methods of making and using them

ABSTRACT

The present invention relates to oligooxopiperazines and their use. Methods for preparing oligooxopiperazines are also disclosed.

This application is a continuation of U.S. patent application Ser. No.14/304,304, filed Jun. 13, 2014, which is a continuation of U.S. patentapplication Ser. No. 12/917,176, filed Nov. 1, 2010, now U.S. Pat. No.8,791,121, which claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 61/373,108, filed Aug. 12, 2010, each of which arehereby incorporated by reference in their entirety.

This invention was made with government support under National ScienceFoundation grant number CHE-0848410. The government has certain rightsin this invention.

FIELD OF THE INVENTION

This invention is directed generally to oligooxopiperazines and methodsfor preparing oligooxopiperazines from amino acids.

BACKGROUND OF THE INVENTION

A fundamental limitation of current drug development centers on theinability of traditional pharmaceuticals to target spatially extendedprotein interfaces. The majority of modern pharmaceuticals are smallmolecules that target enzymes or protein receptors with defined pockets.However, in general they cannot target protein—protein interactionsinvolving large contact areas with the required specificity. Examinationof complexes of proteins with other biomolecules reveals that proteinstend to interact with partners via folded sub-domains, in which thebackbone possesses secondary structure. These protein sub-domains rarelyremain structured once excised from the protein; much of their abilityto specifically bind their intended targets is lost because they assumea manifold of shapes rather than the biologically relevant one. Theα-helix is the most prevalent protein secondary structure.

α-Helices play fundamental roles in mediating protein—proteininteractions. Several approaches for stabilizing peptides in helicalconformations or mimicking this conformation with nonnatural oligomershave been described (Henchey et al., Curr. Opin. Chem. Biol. 12: 692-697(2008); Home et al., Acc. Chem. Res. 41: 1399-1408 (2008); Seebach etal., J. Acc. Chem. Res. 41: 1366-1375 (2008); Patgiri et al., Acc. Chem.Res. 41: 1289-1300 (2008); Garner et al., Org. Biomol. Chem. 5:3577-3585 (2007); Goodman et al., Nat. Chem. Biol. 3: 252-262 (2007);Chin et al., Am. Chem. Soc. 123: 2929-2930 (2001)). Examination ofcomplexes of proteins with other biomolecules reveals that often oneface of the helix featuring the i, i+4 and i+7 residues is involved inbinding. Synthetic scaffolds that display protein-like functionality andreproduce the arrangement of key side chains on an α-helix would beinvaluable as inhibitors of selective protein interactions.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an oligooxopiperazine ofFormula I:

wherein:

-   each of R₁, R₂, R₃, and R₄ is independently an amino acid side    chain, H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R    is independently H, an alkyl, or an aryl;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   A is X₁ or C, wherein:    -   X₁ is H, COR′, CO₂R′, CONR′, an alkyl, an aryl, an arylalkyl, a        cycloalkyl, a heteroaryl, a protecting group for protection of        an amine, a targeting moiety, or a tag; wherein R′ is H, an        alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a        targeting moiety, or a tag; and    -   C is a moiety of the formula

-   -    wherein:        -   each X′ is independently H, COR′, CO₂R′, CONR′, N(R″)₂, an            alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a            targeting moiety, or a tag; wherein:            -   R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl,                a heteroaryl, a targeting moiety, or a tag; and            -   each R″ is independently H, an alkyl, an aryl, an                arylalkyl, a cycloalkyl, a heteroaryl, a targeting                moiety, or a tag;        -   R₀ is an amino acid side chain, H, N(R)₂, OR, halogen, an            alkyl, or an aryl; wherein each R is independently H, an            alkyl, or an aryl; and        -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein            each R is independently H, an alkyl, or an aryl; and

-   B is Y or D, wherein:    -   Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, a        cycloalkyl, a heteroaryl, a protecting group for protection of a        carboxylic acid, a targeting moiety, or a tag; wherein:        -   R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a            heteroaryl, a targeting moiety, or a tag; and        -   each R′″ is independently H, CO₂R′, CONR′, an alkyl, an            aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting            moiety, or a tag; and    -   D is a moiety of the formula

-   -    wherein:        -   R₅ is an amino acid side chain, H, N(R)₂, OR, halogen, an            alkyl, or an aryl; wherein each R is independently H, an            alkyl, or an aryl;        -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein            each R is independently H, an alkyl, or an aryl; and        -   E is X₂ or F, wherein:            -   X₂ is H, COR′, CO₂R′, CONR′, an alkyl, an aryl, an                arylalkyl, a cycloalkyl, a heteroaryl, a protecting                group for protection of an amine, a targeting moiety, or                a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl,                a cycloalkyl, a heteroaryl, a targeting moiety, or a                tag; and            -   F is a moiety of the formula

-   -   -   -    wherein:                -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl;                    wherein each R is independently H, an alkyl, or an                    aryl;                -   R₇ is an amino acid side chain; and                -   Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, an                    arylalkyl, a cycloalkyl, a heteroaryl, a protecting                    group for protection of a carboxylic acid, a                    targeting moiety, or a tag; wherein:                -    R′ is H, an alkyl, an aryl, an arylalkyl, a                    cycloalkyl, a heteroaryl, a targeting moiety, or a                    tag; and                -    each R′″ is independently H, CO₂R′, CONR′, an                    alkyl, an aryl, an arylalkyl, a cycloalkyl, a                    heteroaryl, a targeting moiety, or a tag;

-   with the proviso that A and B are not both, respectively, C and D.

The present invention is further directed to pharmaceutical formulationscontaining the oligooxopiperazine of Formula I and methods of inhibitingprotein activity or protein—protein interactions using theoligooxopiperazine of Formula I.

Another aspect of the present invention relates to a method ofinhibiting a protein—protein interaction. This method involvescontacting at least one of the proteins involved in the protein—proteininteraction with an oligooxopiperazine under conditions effective toinhibit the protein—protein interaction. In one embodiment of thisaspect of the present invention, the protein—protein interaction ismediated by a first hot spot amino acid residue and a second hot spotamino acid residue, and the oligooxopiperazine comprises anoligooxopiperazine of Formula II:

wherein:

-   R₁ and R₂ are independently an amino acid side chain, H, N(R)₂, OR,    halogen, an alkyl, or an aryl; wherein each R is independently H, an    alkyl, or an aryl;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   A is X₁ or C, wherein:    -   X₁ is H, COR′, CO₂R′, CONR′, an alkyl, an aryl, an arylalkyl, a        cycloalkyl, a heteroaryl, a protecting group for protection of        an amine, a targeting moiety, or a tag; wherein R′ is H, an        alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a        targeting moiety, or a tag; and    -   C is a moiety of the formula

-   -    wherein:        -   each X′ is independently H, COR′, CO₂R′, CONR′, N(R″)₂, an            alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a            targeting moiety, or a tag; wherein:        -   R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a            heteroaryl, a targeting moiety, or a tag; and        -   each R″ is independently H, CO₂R′, CONR′, an alkyl, an aryl,            an arylalkyl, a cycloalkyl, a heteroaryl, a targeting            moiety, or a tag;        -   R₀ is an amino acid side chain, H, N(R)₂, OR, halogen, an            alkyl, or an aryl; wherein each R is independently H, an            alkyl, or an aryl; and        -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein            each R is independently H, an alkyl, or an aryl; and

-   Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, a    cycloalkyl, a heteroaryl, a protecting group for protection of a    carboxylic acid, a targeting moiety, or a tag; wherein:    -   R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a        heteroaryl, a targeting moiety, or a tag; and    -   each R′″ is independently H, CO₂R′, CONR′, an alkyl, an aryl, an        arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a        tag.

The present invention is further directed to methods of solid phase andsolution phase synthesis of the oligooxopiperazines of the presentinvention.

A fundamental limitation of current drug development centers on theinability of traditional pharmaceuticals to target spatially extendedprotein surfaces. The intrinsic conformational and chemicalinstabilities of peptides limit their potential as reagents in molecularbiology and drug discovery. Accordingly, there is a need to developnonpeptidic oligomers that display protein-like side chains asalternatives to peptides and have superior pharmacological properties.The present invention describes the design and synthesis of nonpeptidicoxopiperazine oligomers that are non-aromatic helix mimetics that areeasily synthesized from α-amino acids. These scaffolds present chiralbackbones, as compared to the aromatic templates, that are moreeffective in discriminating between chiral protein pockets. Importantly,because the oligooxopiperazines of the present invention are obtained bylinking neighboring amide nitrogen atoms in peptides with ethylenebridges, the amide bond, that may be the chief culprit leading to thepoor cellular uptake of peptides, is removed. Molecular modelingstudies, 2D NMR, and circular dichroism provide strong support for thedesign features of the oligooxopiperazines described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H depict suitable methods of coupling and cyclizing amino acidresidues using various alkylating agents. FIGS. 1A-1B show the steps ofcoupling and cyclizing amino acid residues using X—CH₂—CH═CH (designatedas alkylating agent A) in the solid phase (designated Sd) or solutionphase (designated Sn) synthesis of the oligooxopiperazines of thepresent invention. FIGS. 1C-1D show the steps of coupling and cyclizingamino acid residues using X—CH₂—CH(OR₁₁)₂ (designated as alkylatingagent B) in the solid phase (designated Sd) or solution phase(designated Sn) synthesis schemes of the present invention. FIGS. 1E-1Fshow the steps of coupling and cyclizing amino acid residues usingX—(CH₂)₂—X (designated as alkylating agent C) in the solid phase(designated Sd) or solution phase (designated Sn) synthesis schemes ofthe present invention. FIGS. 1G-1H shows the steps of coupling andcyclizing amino acid residues using X—(CH₂)₂—OH (designated asalkylating agent D) in the solid phase (designated Sd) or solution phase(designated Sn) synthesis schemes of the present invention.

FIGS. 2A-2C illustrate the design and predicted structure of aminoacid-derived oligooxopiperazines. The oligooxopiperazines are obtainedby linking neighboring amide nitrogen atoms in peptides with ethylenebridges as depicted in FIG. 2A. FIG. 2B shows an 8mer canonical α-helixwith side chain residues depicted as dark grey spheres (left). Thepredicted structure of an oligooxopiperazine dimer with side chainresidues depicted as light grey spheres (FIG. 2B, right) and an overlayof the piperazine dimer and the α-helix (FIG. 2B, center) is also shown.FIG. 2C (left) and FIG. 2C (right) show a top-down view of thestructures shown in FIG. 2B (left) and FIG. 2B (center), respectively.

FIGS. 3A-3C depict the rotatable bonds and favored geometries of anoligooxopiperazine dimer. The rotatable bonds (i.e., φ, ψ, and ω) of anoligooxopiperazine dimer are show in FIG. 3A. The favored chair andamide bond geometries are shown in FIGS. 3B and 3C, respectively. Thevalues were calculated with Macromodel MMFF force field in chloroform.

FIGS. 4A-4B show three oligooxopiperazine helix mimetics of the presentinvention (FIG. 4A; oxopiperazine 1a, 1b, and 1c) and their synthesisvia reductive amination (FIG. 4B). Synthesis of dimers 1a-c: (a) O₃, (b)Me₂S, (c) TFA and triethylsilane. Combined yield for steps a-c: 3a, 81%;3b, 80%; 3c, 85%; (d) Boc₂O: 4a, 98%; 4b, 94%; 4c, 97%; (e) LiOH₃, DCC,HOBt: 1a, 73%; 1b, 70%; 1c, 71%. a: R¹═CH₂CH(CH₃)₂, R²═CH₃. b: R¹═CH₂Ph,R²═(CH₂)₄NHCbz. c: R¹═CH₂CH(CH₃)₂, R²═CH₂CH(CH₃)₂.

FIGS. 5A-5C show the solution conformation and thermal stabilities ofoligooxopiperazines 1a, 1b, and 1c shown in FIG. 4A. The circulardichroism (CD) spectra of oxopiperazines 1a-1c in acetonitrile andmethanol is depicted in FIGS. 5A and 5C, respectively. The effect oftemperature on the stability of compounds 1a-1c is shown in FIG. 5B.

FIGS. 6A-6B show a cross-section of the NOESY spectra ofoligooxopiperazine 1a in CDCl₃ (FIG. 6A) and an overlay of key NOEs onthe predicted oligooxopiperazine conformation (FIG. 6B) (Side chaingroups not shown for clarity.)

FIGS. 7A-7B are graphs showing the low energy φ (FIG. 7A) and ψ (FIG.7B) angles for oligooxopiperazine dimer 30 calculated using themacromodel “dihedral drive” function.

FIGS. 8A-8D depict a 10mer alpha helix and the predicted structure of anoligooxopiperazine trimer. The 10mer alpha helix of FIG. 8A displays iand i+1, and i and i+4 distances. FIG. 8B shows the predicted structureof an oligooxopiperazine trimer. An overlay of the trimer and theα-helix (gray stick model) is shown in FIG. 8C. The spheres representamino acid side chains. FIG. 8D illustrates the numbering of side chainresidues on the oligooxopiperazine trimer.

FIGS. 9A-9D show the design and structure of model oligooxopiperazinedimers A-C (FIGS. 9B-9D) and a model oligooxopiperazine trimer (FIG. 9A)of the present invention. An overlay of the predicted structure of eachmodel oligooxopiperazine and its target α-helix is also shown.

FIGS. 10A-10C show oligooxopiperazine 38 of the present inventiondesigned to target the p53 transactivation domain, which adopts ahelical conformation to target Mdm2. Three key hydrophobic residues ofp53 (F19, W23, and L26) bind in the Mdm2 pocket as depicted in FIG. 10A.FIG. 10B shows an overlay of oligooxopiperazine 38 and the p53 helix.FIG. 10C shows the structures of oligooxopiperazine 38 (FIG. 10C; left),and the negative control oligooxopiperazine 39 (FIG. 10C; right), whichlacks the key tryptophan residue.

FIGS. 11A1-11AA2 contain a table of α-helices involved in modulatingprotein—protein interactions that are suitable targets foroligooxopiperazines design. The table sets forth the α-helices by, interalia, their RSC Protein Data Bank (an online database that includesproteins involved in protein—protein interactions; “PDB”) code (columnA), title (column D), function (column E), the chains in theprotein—protein complex featuring a helix at the interface (column B),and the chain containing the candidate helix to be mimicked (column C).Also shown in the table are the number of hot spot residues in the helix(column J), the relative position of the hot spot residues within thechain (column K) and within the helix (column L), the length of thecandidate helix to be mimicked (column N), the first (column 0) and last(column P) residue of the helix to be mimicked, and the amino acidsequence of the helix to be mimicked (column Q).

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is directed to anoligooxopiperazine of Formula I:

wherein:

-   each of R₁, R₂, R₃, and R₄ is independently an amino acid side    chain, H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R    is independently H, an alkyl, or an aryl;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   A is X₁ or C, wherein:    -   X₁ is H, COR′, CO₂R′, CONR, an alkyl, an aryl, an arylalkyl, a        cycloalkyl, a heteroaryl, a protecting group for protection of        an amine, a targeting moiety, or a tag; wherein R′ is H, an        alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a        targeting moiety, or a tag; and    -   C is a moiety of the formula

-   -    wherein:        -   each X′ is independently H, COR′, CO₂R′, CONR′, N(R″)₂, an            alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a            targeting moiety, or a tag; wherein:            -   R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl,                a heteroaryl, a targeting moiety, or a tag; and            -   each R″ is independently H, CO₂R′, CONR′, an alkyl, an                aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a                targeting moiety, or a tag;        -   R₀ is an amino acid side chain, H, N(R)₂, OR, halogen, an            alkyl, or an aryl; wherein each R is independently H, an            alkyl, or an aryl; and        -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein            each R is independently H, an alkyl, or an aryl; and

-   B is Y or D, wherein:    -   Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, a        cycloalkyl, a heteroaryl, a protecting group for protection of a        carboxylic acid, a targeting moiety, or a tag; wherein:        -   R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a            heteroaryl, a targeting moiety, or a tag; and        -   each R′″ is independently H, CO₂R′, CONR′, an alkyl, an            aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting            moiety, or a tag; and    -   D is a moiety of the formula

-   -    wherein:        -   R₅ is an amino acid side chain, H, N(R)₂, OR, halogen, an            alkyl, or an aryl; wherein each R is independently H, an            alkyl, or an aryl;        -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein            each R is independently H, an alkyl, or an aryl; and        -   E is X₂ or F, wherein:            -   X₂ is H, COR′, CO₂R′, CONR′, an alkyl, an aryl, an                arylalkyl, a cycloalkyl, a heteroaryl, a protecting                group for protection of an amine, a targeting moiety, or                a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl,                a cycloalkyl, a heteroaryl, a targeting moiety, or a                tag; and            -   F is a moiety of the formula

-   -   -   -    wherein:                -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl;                    wherein each R is independently H, an alkyl, or an                    aryl;                -   R₇ is an amino acid side chain; and                -   Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, an                    arylalkyl, a cycloalkyl, a heteroaryl, a protecting                    group for protection of a carboxylic acid, a                    targeting moiety, or a tag; wherein:                -    R′ is H, an alkyl, an aryl, an arylalkyl, a                    cycloalkyl, a heteroaryl, a targeting moiety, or a                    tag; and                -    each R′″ is independently H, CO₂R′, CONR′, an                    alkyl, an aryl, an arylalkyl, a cycloalkyl, a                    heteroaryl, a targeting moiety, or a tag;

-   with the proviso that A and B are not both, respectively, C and D.

Amino acid side chains according to this and all aspects of the presentinvention can be any amino acid side chain—from natural or nonnaturalamino acids—including alpha amino acids, beta amino acids, gamma aminoacids, L-amino acids, and D-amino acids.

As used herein, the term “alkyl” means an aliphatic hydrocarbon groupwhich may be straight or branched having about 1 to about 6 carbon atomsin the chain. Branched means that one or more lower alkyl groups such asmethyl, ethyl or propyl are attached to a linear alkyl chain. Exemplaryalkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, n-pentyl, and 3-pentyl.

As used herein, “cycloalkyl” refers to a non-aromatic saturated orunsaturated mono- or polycyclic ring system which may contain 3 to 6carbon atoms, and which may include at least one double bond. Exemplarycycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, anti-bicyclopropane, or syn-bicyclopropane.

As used herein, the term “aryl” refers to an aromatic monocyclic orpolycyclic ring system containing from 6 to 19 carbon atoms, where thering system may be optionally substituted. Aryl groups of the presentinvention include, but are not limited to, groups such as phenyl,naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl,triphenylenyl, chrysenyl, and naphthacenyl.

The term “arylalkyl” refers to a radical of the formula —R^(a)R^(b)where R^(a) is an alkyl radical as defined above and R^(b) is an arylradical as defined above. The alkyl radical and the cycloalkyl radicalmay be optionally substituted as defined above.

As used herein, “heteroaryl” refers to an aromatic ring radical whichconsists of carbon atoms and from one to five heteroatoms selected fromthe group consisting of nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups include, without limitation, pyrrolyl, pyrazolyl,imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl, furopyrrolyl,indolyl, azaindolyl, isoindolyl, indolinyl, indolizinyl, indazolyl,benzimidazolyl, imidazopyridinyl, benzotriazolyl, benzoxazolyl,benzoxadiazolyl, benzothiazolyl, pyrazolopyridinyl, triazolopyridinyl,thienopyridinyl, benzothiadiazolyl, benzofuyl, benzothiophenyl,quinolinyl, isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl,cinnolinyl, quinazolinyl, quinolizilinyl, phthalazinyl, benzotriazinyl,chromenyl, naphthyridinyl, acrydinyl, phenanzinyl, phenothiazinyl,phenoxazinyl, pteridinyl, and purinyl. Additional heteroaryls aredescribed in COMPREHENSIVE HETEROCYCLIC CHEMISTRY: THE STRUCTURE,REACTIONS, SYNTHESIS AND USE OF HETEROCYCLIC COMPOUNDS (Katritzky et al.eds., 1984), which is hereby incorporated by reference in its entirety.

The oligooxopiperazines of Formula I may comprise a protecting groupthat is suitable for the protection of an amine or a carboxylic acid.Such protecting groups function primarily to protect or mask thereactivity of functional groups. Protecting groups that are suitable forthe protection of an amine group are well known in the art, includingwithout limitation, carbamates, amides, N-alkyl and N-aryl amines, iminederivatives, enamine derivatives, and N-hetero atom derivatives asdescribed by THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS INORGANIC SYNTHESIS 494-615 (1999), which is hereby incorporated byreference in its entirety. Protecting groups that are suitable for theprotection of a carboxylic acid are also well known in the art. Suitablecarboxylic acid protecting groups include, without limitation, esters(e.g., substituted methyl esters, 2-substituted ethyl esters,2,6-dialkylphenyl esters, substituted benzyl esters, silyl esters, andstannyl esters), amides, and hydrazides as described by THEODORA W.GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS372-450 (1999), which is hereby incorporated by reference in itsentirety. Methods of protecting and deprotecting amine and carboxylicacids vary depending on the chosen protecting group; however, thesemethods are well known in the art and described in THEODORA W. GREENE &PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 372-450 and494-615 (1999), which is hereby incorporated by reference in itsentirety.

A “tag” as used herein includes any labeling moiety that facilitates thedetection, quantitation, separation, and/or purification of theoligooxopiperazine of the present invention. Suitable tags includepurification tags, radioactive or fluorescent labels, and enzymatictags.

Purification tags, such as poly-histidine (His₆₋), aglutathione-S-transferase (GST-), or maltose-binding protein (MBP-), canassist in oligomer purification or separation but can later be removed,i.e., cleaved from the oligooxopiperazine following recovery.Protease-specific cleavage sites can be used to facilitate the removalof the purification tag. The desired oligooxopiperazine product can bepurified further to remove the cleaved purification tags.

Other suitable tags include radioactive labels, such as, ¹²⁵I, ¹³¹I,¹¹¹In, or ⁹⁹TC. Methods of radiolabeling compounds, are known in the artand described in U.S. Pat. No. 5,830,431 to Srinivasan et al., which ishereby incorporated by reference in its entirety. Radioactivity isdetected and quantified using a scintillation counter orautoradiography. Alternatively, the oligooxopiperazine can be conjugatedto a fluorescent tag. Suitable fluorescent tags include, withoutlimitation, chelates (europium chelates), fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, Lissamine,phycoerythrin and Texas Red. The fluorescent labels can be conjugated tothe oligooxopiperazine using techniques disclosed in CURRENT PROTOCOLSIN IMMUNOLOGY (Coligen et al. eds., 1991), which is hereby incorporatedby reference in its entirety. Fluorescence can be detected andquantified using a fluorometer.

Enzymatic tags generally catalyze a chemical alteration of a chromogenicsubstrate which can be measured using various techniques. For example,the enzyme may catalyze a color change in a substrate, which can bemeasured spectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Examples of suitableenzymatic tags include luciferases (e.g., firefly luciferase andbacterial luciferase; see e.g., U.S. Pat. No. 4,737,456 to Weng et al.,which is hereby incorporated by reference in its entirety), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases(e.g., horseradish peroxidase), alkaline phosphatase, 0-galactosidase,glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (e.g., uricase and xanthine oxidase), lactoperoxidase,microperoxidase, and the like. Techniques for conjugating enzymes toproteins and peptides are described in O'Sullivan et al., Methods forthe Preparation of Enzyme—Antibody Conjugates for Use in EnzymeImmunoassay, in METHODS IN ENZYMOLOGY 147-66 (Langone et al. eds.,1981), which is hereby incorporated by reference in its entirety. Suchtags may be particularly useful for detecting inhibition ofprotein—protein interactions using the oligooxopiperazine of the presentinvention, as described more fully, infra.

A targeting moiety according to the present invention functions to (i)promote the cellular uptake of the oligooxopiperazine, (ii) target theoligooxopiperazine to a particular cell or tissue type (e.g., signalingpeptide sequence), or (iii) target the oligooxopiperazine to a specificsub-cellular localization after cellular uptake (e.g., transport peptidesequence).

To promote the cellular uptake of an oligooxopiperazine of the presentinvention, the targeting moiety may be a cell penetrating peptide (CPP).CPPs translocate across the plasma membrane of eukaryotic cells by aseemingly energy-independent pathway and have been used successfully forintracellular delivery of macromolecules, including antibodies,peptides, proteins, and nucleic acids, with molecular weights severaltimes greater than their own. Several commonly used CPPs, includingpolyarginines, transportant, protamine, maurocalcine, and M918, aresuitable targeting moieties for use in the present invention and arewell known in the art (see Stewart et al., “Cell-Penetrating Peptides asDelivery Vehicles for Biology and Medicine,” Organic Biomolecular Chem6:2242-2255 (2008), which is hereby incorporated by reference in itsentirety). Additionally, methods of making CPP are described in U.S.Patent Application Publication No. 20080234183 to Hallbrink et al.,which is hereby incorporated by reference in its entirety.

Another suitable targeting moiety useful for enhancing the cellularuptake of the oligooxopiperazine is an “importation competent” signalpeptide as disclosed by U.S. Pat. No. 6,043,339 to Lin et al., which ishereby incorporated by reference in its entirety. An importationcompetent signal peptides is generally about 10 to about 50 amino acidresidues in length, typically hydrophobic residues, that render theoligooxopiperazine capable of penetrating through the cell membrane fromoutside the cell to the interior of the cell. An exemplary importationcompetent signal peptide includes the signal peptide from Kaposifibroblast growth factor (see U.S. Pat. No. 6,043,339 to Lin et al.,which is hereby incorporated by reference in its entirety). Othersuitable peptide sequences can be selected from the SIGPEP database (seevon Heijne G., “SIGPEP: A Sequence Database for Secretory SignalPeptides,” Protein Seq. Data Anal. 1(1):41-42 (1987), which is herebyincorporated by reference in its entirety).

Another suitable targeting moiety is a signal peptide sequence capableof targeting the oligooxopiperazine to a particular tissue or cell type.The signaling peptide can include at least a portion of a ligand bindingprotein. Suitable ligand binding proteins include high-affinity antibodyfragments (e.g., Fab, Fab′ and F(ab′)₂), single-chain Fv antibodyfragments), nanobodies or nanobody fragments, fluorobodies, or aptamers.Other ligand binding proteins include biotin-binding proteins,lipid-binding proteins, periplasmic binding proteins, lectins, serumalbumins, enzymes, phosphate and sulfate binding proteins,immunophilins, metallothionein, or various other receptor proteins. Forcell specific targeting, the signaling peptide is preferably a ligandbinding domain of a cell specific membrane receptor. Thus, when themodified oligooxopiperazine is delivered intravenously or otherwiseintroduced into blood or lymph, the oligooxopiperazine will adsorb tothe targeted cell, and the targeted cell will internalize theoligooxopiperazine. For example, if the target cell is a cancer cell,the oligooxopiperazine may be conjugated to an anti-C3B(I) antibody asdisclosed by U.S. Pat. No. 6,572,856 to Taylor et al., which is herebyincorporated by reference in its entirety. Alternatively, theoligooxopiperazine may be conjugated to an alphafeto protein receptor asdisclosed by U.S. Pat. No. 6,514,685 to Moro, or to a monoclonal GAHantibody as disclosed by U.S. Pat. No. 5,837,845 to Hosokawa, which arehereby incorporated by reference in their entirety. For targeting anoligooxopiperazine to a cardiac cell, the oligooxopiperazine may beconjugated to an antibody recognizing elastin microfibril interfacer(EMILIN2) (Van Hoof et al., “Identification of Cell Surface forAntibody-Based Selection of Human Embryonic Stem Cell-DerivedCardiomyocytes,” J Proteom Res 9:1610-18 (2010), which is herebyincorporated by reference in its entirety), cardiac troponin I,connexin-43, or any cardiac cell-surface membrane receptor that is knownin the art. For targeting an oligooxopiperazine to a hepatic cell, thesignaling peptide may include a ligand domain specific to thehepatocyte-specific asialoglycoprotein receptor. Methods of preparingsuch chimeric proteins and peptides are described in U.S. Pat. No.5,817,789 to Heartlein et al., which is hereby incorporated by referencein its entirety.

Another suitable targeting moiety is a transport peptide that directsintracellular compartmentalization of the oligooxopiperazine once it isinternalized by a target cell or tissue. For example, if the proteinactivity or protein—protein interaction that is sought to be inhibitedoccurs in the endoplasmic reticulum (ER), the oligooxopiperazine can beconjugated to an ER transport peptide sequence. A number of such signalpeptides are known in the art, including the signal peptideMMSFVSLLLVGILFYATEAEQLTKCEVFQ (SEQ ID NO:182). Other suitable ER signalpeptides include the N-terminus endoplasmic reticulum targeting sequenceof the enzyme 17β-hydroxysteroid dehydrogenase type 11 (Horiguchi etal., “Identification and Characterization of the ER/LipidDroplet-Targeting Sequence in 17β-hydroxysteroid Dehydrogenase Type 11,”Arch. Biochem. Biophys. 479(2):121-30 (2008), which is herebyincorporated by reference in its entirety), or any of the ER signalingpeptides (including the nucleic acid sequences encoding the ER signalpeptides) disclosed in U.S. Patent Publication No. 20080250515 to Reedet al., which is hereby incorporated by reference in its entirety.Additionally, the oligooxopiperazine of the present invention cancontain an ER retention signal, such as the retention signal KEDL (SEQID NO:183). Methods of modifying the oligooxopiperazines of the presentinvention to incorporate transport peptides for localization of theoligomers to the ER can be carried out as described in U.S. PatentPublication No. 20080250515 to Reed et al., which is hereby incorporatedby reference in its entirety.

If the protein activity or protein—protein interaction that is sought tobe inhibited occurs in the nucleus, the oligooxopiperazine can include anuclear localization transport signal. Suitable nuclear transportpeptide sequences are known in the art, including the nuclear transportpeptide PPKKKRKV (SEQ ID NO:184). Other nuclear localization transportsignals include, for example, the nuclear localization sequence ofacidic fibroblast growth factor and the nuclear localization sequence ofthe transcription factor NF-KB p50 as disclosed by U.S. Pat. No.6,043,339 to Lin et al., which is hereby incorporated by reference inits entirety. Other nuclear localization peptide sequences known in theart are also suitable for use in the accordance with this aspect of theinvention.

Suitable transport peptide sequences for targeting to the mitochondriainclude MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:185). Other suitabletransport peptide sequences suitable for selectively targeting theoligooxopiperazine of the present invention to the mitochondria aredisclosed in U.S. Published Patent Application No. 20070161544 to Wipf,which is hereby incorporated by reference in its entirety.

In one embodiment of the present invention, the oligooxopiperazine ofFormula I has a formula of Formula IA:

Exemplary oligooxopiperazine compounds of Formula IA include, withoutlimitation,

where X is H, COCH₃, or any amino acid, and Y is OH, NH₂, OMe, or anyamino acid.

In another embodiment of the present invention, the oligooxopiperazineof Formula I has a formula of Formula IB:

Exemplary oligooxopiperazine compounds of Formula IB include, withoutlimitation,

where X is H, COCH₃, or any amino acid, and Y is OH, NH₂, OMe, or anyamino acid.

In another embodiment of the present invention, the oligooxopiperazineof Formula I has a formula of Formula IC:

Exemplary oligooxopiperazine compounds of Formula IC include, withoutlimitation,

where X is H, COCH₃, or any amino acid, and Y is OH, NH₂, OMe, or anyamino acid.

In a preferred embodiment of the present invention, theoligooxopiperazine of Formula I, including oligooxopiperazines ofFormulas IA, IB, and IC, are designed to mimic an α-helix that isinvolved in a protein—protein interaction. α-Helices involved inmodulating protein—protein interactions that are suitable for mimickingare shown in the table of FIGS. 11A1-11AA2. This table sets forthpredicted targets by, inter alia, their RSC Protein Data Bank (an onlinedatabase that includes proteins involved in protein—proteininteractions; “PDB”) code (column A), title (column D), function (columnE), the chains in the protein—protein complex featuring a helix at theinterface (column B), and the chain containing the candidate helix to bemimicked (column C). Also shown in the table of FIGS. 11A1-11AA2 are thenumber of hot spot residues in the helix (column J), the relativeposition of the hot spot residues within the chain (column K) and withinthe helix (column L), the length of the candidate helix to be mimicked(column N), the first (column O) and last (column P) residue of thehelix to be mimicked, and the amino acid sequence of the helix to bemimicked (column Q). Additional α-helices suitable for mimicking aredisclosed in Jochim et al., “Assessment of Helical Interfaces inProtein—Protein Interactions,” Mol. Biosyst. 5(9):924-26 (2009), whichis hereby incorporated by reference in its entirety, which describes theidentification and classification of over 2,500 helical interfaceprotein—protein interactions and the hot spot residues involved in theseinteractions.

Oligooxopiperazines of the present invention that are designed to mimican α-helix of a protein, e.g., an α-helix involved in a protein—proteininteraction, can be designed to mimic every side chain of the α-helix.Alternatively, if the hot spot residues of the α-helix are known, theoligooxopiperazine can be designed to mimic only the hot spot residues,in which case the remaining oligooxopiperazine side groups can be anyside group that does not interfere with the oligooxopiperazine'sfunction.

In accordance with this embodiment of the present invention, R₁, R₂, R₄,and R₅ of the oligooxopiperazine of Formula IA can mimic the amino acidside chain of, respectively, residues i, i+4, i+6, and i+7, of theα-helix. Suitable oligooxopiperazines of Formula IA that mimic anα-helix involved in a protein—protein interaction include, withoutlimitation,

where X is H, COCH₃, or any amino acid, and Y is OH, NH₂, OMe, or anyamino acid.

The oligooxopiperazine of Formula IB can also be designed to mimic anα-helix involved in a protein—protein interaction. In one embodiment,R₁, R₂, and R₄ of the oligooxopiperazine of Formula IB can mimic theamino acid side chain of, respectively, residues i, i+4, and i+7, of theα-helix. Such suitable oligooxopiperazines of Formula IB include,without limitation,

where X is H, COCH₃, or any amino acid, and Y is OH, NH₂, OMe, or anyamino acid. Alternatively, R₁, R₂, and R₄ can mimic the amino acid sidechain of, respectively, residues i, i+4, and i+6 of the α-helix. Suchsuitable oligooxopiperazines of Formula IB include, without limitation,

where X is H, COCH₃, or any amino acid, and Y is OH, NH₂, OMe, or anyamino acid.

The oligooxopiperazine of Formula IC can also be designed to mimic anα-helix involved in protein—protein interactions. For example, R₀, R₁,R₂, R₃, and R₄ of Formula IC can mimic the amino acid side chain of,respectively, residues i, i+2, i+3, i+4, and i+7 of the α-helix.Suitable oligooxopiperazines of Formula IC that mimic an α-helixinvolved in a protein—protein interaction include, without limitation,

where X is H, COCH₃, or any amino acid, and Y is OH, NH₂, OMe, or anyamino acid.

Another aspect of the present invention relates to pharmaceuticalformulations comprising any of the above described oligooxopiperazinesof Formula I, including the oligooxopiperazines of Formulas IA, IB, andIC of the present invention and a pharmaceutically acceptable carrier.Acceptable pharmaceutical carriers include solutions, suspensions,emulsions, excipients, powders, or stabilizers. The carrier should besuitable for the desired mode of delivery.

In addition, the pharmaceutical formulations of the present inventionmay further comprise one or more pharmaceutically acceptable diluents,adjuvants, excipients, or vehicles, such as preserving agents, fillers,disintegrating agents, wetting agents, emulsifying agents, suspendingagents, sweetening agents, flavoring agents, perfuming agents,antibacterial agents, antifungal agents, lubricating agents anddispensing agents, depending on the nature of the mode of administrationand dosage forms. Examples of suspending agents include ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar—agarand tragacanth, or mixtures of these substances. Prevention of theaction of microorganisms can be ensured by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,for example sugars, sodium chloride, and the like. Prolonged absorptionof the injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monosterate andgelatin. Examples of suitable carriers, diluents, solvents, or vehiclesinclude water, ethanol, polyols, suitable mixtures thereof, vegetableoils (such as olive oil), and injectable organic esters such as ethyloleate. Examples of excipients include lactose, milk sugar, sodiumcitrate, calcium carbonate, and dicalcium phosphate. Examples ofdisintegrating agents include starch, alginic acids, and certain complexsilicates. Examples of lubricants include magnesium stearate, sodiumlauryl sulphate, talc, as well as high molecular weight polyethyleneglycols.

Another aspect of the present invention relates to a method ofinhibiting activity of a protein that involves contacting the proteinwith an oligooxopiperazine of the present invention under conditionseffective to inhibit activity of the protein. The oligooxopiperazineaccording to this aspect of the present invention is anoligooxopiperazine of Formula I (e.g, an oligooxopiperazine of FormulaIA, IB, or IC), preferably designed to mimic an α-helix involved in aprotein—protein interaction as described supra.

Another aspect of the present invention relates to a method ofinhibiting a protein—protein interaction that involves contacting atleast one of the proteins involved in the protein—protein interactionwith an oligooxopiperazine under conditions effective to inhibit theprotein—protein interaction. The oligooxopiperazine according to thisaspect of the present invention is an oligooxopiperazine of Formula I(e.g, an oligooxopiperazine of Formula IA, IB, or IC), or, if theprotein—protein interaction is mediated by a first hot spot residue anda second hot spot residue, an oligooxopiperazine Formula II:

wherein:

-   -   R₁ and R₂ are independently an amino acid side chain, H, N(R)₂,        OR, halogen, an alkyl, or an aryl; wherein each R is        independently H, an alkyl, or an aryl;    -   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an        aryl; wherein each R is independently H, an alkyl, or an aryl;    -   A is X₁ or C, wherein:        -   X₁ is H, COR′, CO₂R′, CONR′, an alkyl, an aryl, an            arylalkyl, a cycloalkyl, a heteroaryl, a protecting group            for protection of an amine, a targeting moiety, or a tag;            wherein R′ is H, an alkyl, an aryl, an arylalkyl, a            cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and        -   C is a moiety of the formula

-   -   -    wherein:            -   each X′ is independently H, COR′, CO₂R′, CONR′, N(R″)₂,                an alkyl, an aryl, an arylalkyl, a cycloalkyl, a                heteroaryl, a targeting moiety, or a tag; wherein:                -   R′ is H, an alkyl, an aryl, an arylalkyl, a                    cycloalkyl, a heteroaryl, a targeting moiety, or a                    tag; and                -   each R″ is independently H, CO₂R′, CONR′, an alkyl,                    an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a                    targeting moiety, or a tag;            -   R₀ is an amino acid side chain, H, N(R)₂, OR, halogen,                an alkyl, or an aryl; wherein each R is independently H,                an alkyl, or an aryl; and            -   R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl;                wherein each R is independently H, an alkyl, or an aryl;                and

    -   Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, a        cycloalkyl, a heteroaryl, a protecting group for protection of a        carboxylic acid, a targeting moiety, or a tag; wherein:        -   R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a            heteroaryl, a targeting moiety, or a tag; and        -   each R′″ is independently H, CO₂R′, CONR′, an alkyl, an            aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting            moiety, or a tag.

Preferably, the oligooxopiperazine is designed to mimic an α-helixinvolved in the protein—protein interaction. Oligooxopiperazines ofFormula I can be used to mimic α-helices containing 3-5 hot spotresidues, such as the α-helices identified in FIGS. 11A1-11AA2.Oligooxopiperazines of Formula II can be used to mimic α-helicescontaining only 2 hot spot residues. For example, the first and secondhot spot residues can be, respectively, residues i and i+4 of an alphahelix, and R₁ and R₂ of Formula II can mimic the amino acid side chainof, respectively, residues i and i+4 of the α-helix.

Another aspect of the present invention is directed to a method oftreating a disorder in a subject, where the disorder is mediated by p53.This method involves administering to the subject a pharmaceuticalcomposition containing an oligooxopiperazine that mimics the α-helix ofp53 under conditions effective to treat the disorder. In accordance withthis aspect of the invention, the oligooxopiperazine is preferably anoligooxopiperazine of Formula IA, where R₁, R₂, R₄, and R₅ mimic theamino acid side chain of, respectively, residues i, i+4, i+6, and i+7 ofa p53 α-helix.

In accordance with this aspect of the invention, an oligooxopiperazineof Formula IA that is suitable for treating a disorder mediated by p53in a subject is oligooxopiperazine 38 as described in the Examplesherein and shown in FIG. 10C. Oligooxopiperazines mimicking an α-helixof p53 that disrupt p53 complex formation with, for example, Mdm2, wouldbe suitable for treating cancer. Therapeutic inhibition of p53 is alsosuitable for the treatment of ischemia induced apoptosis, myocardialinfarction, cholestasis, and a variety of neurodegenerative diseasesincluding AID-associated neurodegeneration, stroke, Parkinson's disease,Alzheimer's disease, and Huntington's disease (see Amaral J., “The Roleof p53 in Apoptosis,” Discov. Med. 9(45):145-53 (2010), which is herebyincorporated by reference in its entirety).

Another aspect of the present invention is directed to methods of makingoligooxopiperazines, including the oligooxopiperazines of Formulas IA,IB, and IC. The oligooxopiperazines can be synthesized via solutionphase synthesis, or alternatively, via solid phase synthesis.

Accordingly, one aspect of the present invention is directed to a methodof solid phase synthesis of the oligooxopiperazine of Formula IA. Thismethod of synthesis involves providing a compound of Formula III:

where PG is a protecting group for the protection of an amine; R₈ is anamino acid side chain, H, N(R)₂, OR, halogen, an alkyl, or an aryl,where each R is independently H, an alkyl, or an aryl; and R₉ is —O-Resor —NH-Res, where Res is a solid phase peptide synthesis resin. Thismethod further involves providing a compound of Formula IV₁:

PG is a protecting group for the protection of an amine and where R₁₀ is—OH or a halide. The compound of Formula III is reacted with a firstalkylating agent and the compound of Formula IV₁ under conditionseffective to produce a compound of Formula V:

If necessary, —CR₆R₈—CO—R₉ in the compound of Formula V can be convertedto E of Formula IA using standard methods known in the art. In addition,if necessary the N-terminal hydrogen in the compound of Formula V can beconverted to X₁ of Formula IA. As will be appreciated by one of skill inthe art, according to this and all aspects of the present invention thatcall for converting a first moiety to a second moiety, said convertingcan be carried out, for example, by chemically transforming the firstmoiety to the second moiety or by entirely replacing the first moietywith the second moiety.

This and other synthesis methods described herein include the use ofindividual amino acid residues. Typically, individual amino acidresidues are obtained protected at the N-terminal and unprotected at theC-terminal. The C-terminal can then be protected using standard methodsknown in the art (see e.g., THEODORA W. GREENE & PETER G. M. WUTS,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 372-450 and 494-615 (1999), whichis hereby incorporated by reference in its entirety). If desired, theN-terminal protecting group in the amino acid residue can be replacedwith a different amino protecting group for use in these methods.

In accordance with this and subsequent solid phase synthesis embodimentsof the invention, solid phase peptide synthesis resins suitable for useinclude, without limitation, polystyrene resins, polyamide resins, PEGhybrid polystyrene resins, and PEG-based resins as described in FlukaChemie GmbH, “Resins for Solid—Phase Peptide Synthesis,” ChemFiles3(4):5-6 (2003), which is hereby incorporated by reference in itsentirety.

In this and all synthesis methods described herein, suitable protectinggroups for the protection of an amine include any of those describedsupra. Exemplary protecting groups include Boc, Cbz, Ns, and Fmoc.Likewise, in all synthesis methods described herein, suitable protectinggroups for the protection of a carboxylic acid include any of thosedescribed supra.

In accordance with this and all aspects of the present invention,suitable halides include Br, Cl, and F. Preferably, the halide is Br.

In all the synthesis methods described herein involving the use of analkylating agent, suitable alkylating agents include those selected fromthe group consisting of X—CH₂—CH═CH, X—CH₂—CH(OR₁₁)₂, X—(CH₂)₂—X, andX—(CH₂)₂—OH, wherein each X is independently a leaving group and eachR₁₁ is independently an alkyl (e.g., halogens, OMs, or OTs). Suitablemethods of using the above alkylating agents are depicted in FIGS. 1A-1Hand are described infra. Where more than one alkylating step is calledfor, the alkylating agent for each step can be the same or different.

In accordance with the above method of making the oligooxopiperazine ofFormula IA, the compound of Formula III can be provided by providing acompound of Formula VI:

and a compound of Formula IV₂:

The compound of Formula VI is reacted with the compound of Formula IV₂under conditions effective to produce a compound of Formula III usingmethods that will be apparent to one of ordinary skill in the art.

The compound of Formula VI above can be provided by providing a compoundof Formula VII:

and a compound of Formula IV₃:

The compound of Formula VII is reacted with a second alkylating agentand the compound of Formula IV₃ under conditions effective to produce acompound of Formula VI using methods that will be apparent to one ofordinary skill in the art.

The compound of Formula VII above can be provided by providing andreacting a compound of Formula VIII:

and a compound of Formula IV₄:

under conditions effective to produce a compound of Formula VII usingmethods that will be apparent to one of ordinary skill in the art.

The compound of Formula VIII above can be provided by providing acompound of Formula IX:

and a compound of Formula IV₅:

The compound of Formula IX is reacted with a third alkylating agent andthe compound of Formula IV₅ under conditions effective to produce acompound of Formula VIII using methods that will be apparent to one ofordinary skill in the art.

Another aspect of the present invention is directed to the solid phasesynthesis of the oligooxopiperazines of Formula IB and IC. This methodof synthesis involves providing a compound of Formula VII′:

where PG is a protecting group for the protection of an amine, and R₉ is—O-Res or —NH-Res. The method further involves providing a compound ofFormula

where PG is a protecting group for the protection of an amine and R₁₀ is—OH or a halide. The compound of Formula VII′ is reacted with a firstalkylating agent and the compound of Formula IV₁ under conditionseffective to produce a compound of Formula VI′:

using methods that will be apparent to one of ordinary skill in the art.

If necessary, —R₉ of Formula VI′ can be converted to Y using standardmethods known in the art. Further, when synthesizing anoligooxopiperazine of Formula 1B, if necessary, the N-terminal hydrogenin the compound of Formula VI′ can be converted to X₁ using standardmethods known in the art; when synthesizing an oligooxopiperazine ofFormula IC, the N-terminal hydrogen in the compound of Formula VI′ canbe converted to a moiety of formula

using standard methods.

In accordance with the above method of making the oligooxopiperazines ofFormulas IB and IC, the compound of Formula VII′ can be provided byproviding a compound of Formula VIII′:

and providing a compound of Formula IV₂:

The compound of Formula VIII′ is reacted with the compound of FormulaIV₂ under conditions effective to produce a compound of Formula VII′using methods that will be apparent to one of ordinary skill in the art.

A compound of Formula VIII′ can be provided by providing a compound ofFormula IX′:

and providing a compound of Formula IV₃:

The compound of Formula IX′ is reacted with a second alkylating agentand the compound of Formula IV₃ under conditions effective to produce acompound of Formula VIII′ using methods that will be apparent to one ofordinary skill in the art.

Another aspect of the present invention is directed to a method ofsolution phase synthesis of the oligooxopiperazines of Formula IA. Thismethod of synthesis involves providing a compound of Formula X:

where PG₁ is a protecting group for the protection of an amine and R₁₀is —OH or a halide, and providing a compound of Formula XI_(5/8):

where PG₂ is a protecting group for the protection of a carboxylic acid;and R₈ is an amino acid side chain, H, N(R)₂, OR, halogen, an alkyl, oran aryl, where each R is independently H, an alkyl, or an aryl. Thecompound of Formula X is reacted with the compound of Formula XI_(5/8)under conditions effective to produce a compound of Formula XII:

If necessary, —CR₆R₈—CO—PG₂ in the compound of Formula XII can beconverted to E of Formula IA using standard methods known in the art.Additionally, if necessary PG₁ in the compound of Formula XII can beconverted to X₁ of Formula IA using standard methods known in the art.

The compound of Formula X above can be provided by providing a compoundof Formula X′:

and converting PG₁ of Formula X′ to hydrogen and converting PG₂ ofFormula X′ to R₁₀. Methods for removing protecting groups are well knownin the art (see e.g., THEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVEGROUPS IN ORGANIC SYNTHESIS 372-450 and 494-615 (1999), which is herebyincorporated by reference in its entirety).

The compound of Formula X′ above can be provided by providing a compoundof Formula XIII.

and providing a compound of Formula XI_(3/4):

The compound of Formula XIII is reacted with the compound of FormulaXI_(3/4) under conditions effective to produce a compound of Formula X′using methods that will be apparent to one of ordinary skill in the art.

The compound of Formula XIII above can be provided by providing acompound of Formula XI_(1/2):

and reacting it with a protecting group under conditions effective toproduce a compound of Formula XIII using methods that will be apparentto one of ordinary skill in the art. Suitable methods for addingprotecting groups are well known in the art (see e.g., THEODORA W.GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS372-450 and 494-615 (1999), which is hereby incorporated by reference inits entirety).

The compound of Formula XI_(1/2) can be provided by providing a compoundof Formula XIV:

and providing a compound of Formula XV:

The compound of Formula XIV is reacted with an alkylating agent and thecompound of Formula XV under conditions effective to produce a compoundof Formula XI_(1/2).

Another aspect of the present invention is directed to the solutionphase synthesis of the oligooxopiperazines of Formula IB and IC. Thismethod of synthesis involves providing a compound of Formula XIII:

where PG₁ is a protecting group for the protection of an amine and R₁₀is —OH or a halide. Suitable methods of making the compound of FormulaXIII are described supra. A compound of Formula XI_(3/4):

where PG₂ is a protecting group for the protection of a carboxylic acid,is also provided. The compound of Formula XIII is reacted with thecompound of Formula XI_(3/4) under conditions effective to produce acompound of Formula X′:

If necessary, PG₂ in the compound of Formula X′ can be converted to Yusing standard methods known in the art. Further, when synthesizing theoligooxopiperazine of Formula IB, if necessary, PG₁ in the compound ofFormula X′ can be converted to X₁ using standard methods; whensynthesizing the oligooxopiperazine of Formula IC, PG₁ in the compoundof Formula X′ can be converted to a moiety of formula

using standard methods.

The above described solid phase and solution phase methods ofoligooxopiperazine synthesis sometimes call for reacting compounds withan alkylating agent. The alkylating agent is used to facilitate couplingand cyclization of the oligooxopiperazine. Suitable methods of couplingand cyclization using the exemplary alkylating agents disclosed hereininclude the methods shown in FIGS. 1A-1H.

In particular, FIGS. 1A-1B depict coupling and cyclization usingX—CH₂—CH═CH as the alkylating agent (alkylating agent A) in solid phase(Sd; left) and solution phase (Sn; right) methods of synthesis. Step Ainvolves the alkylation of the amino acid residue (1_(ASd) or 1_(ASn)).PG₃, which is a protecting group for the protection of an amine, allowsthe alkylating agent to react with the hydrogen on the amine. Ns is apreferred protecting group for this purpose. PG₃ can then be replacedwith hydrogen to facilitate coupling with another residue. Typically, amild base is used during alkylation to facilitate hydrogen removal.Suitable bases include triethylamine, N,N-diisopropylethylamine,1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 2,4,6-trimethylpyridine,potassium carbonate, and cesium carbonate.

Step B involves the coupling of a second amino acid residue (2_(ASd) or2_(ASn)) to the alkylated amino acid residue (1′_(ASd) or 1′_(ASn)). InStep C, the coupled amino acid residues (3_(ASd) or 3_(ASn)) arecyclized upon the simultaneous or sequential addition of an oxidizingagent, an acid, and a hydride donor. The oxidizing agent, preferablyozone, converts the allyl to an aldehyde. The acid is one that removesthe protecting group to provide for cyclization with the aldehyde.Suitable acids include TFA, HCl, HBr, HCOOH, and CH₃COOH. The hydridedonor ensures that the cyclization reaction takes place in excesshydrogen so the resulting ring is saturated. Suitable hydride donorsinclude triethylsilane and NaBH₃CN.

FIGS. 1C-1D depict coupling and cyclization using X—CH₂—CH(OR₁₁)₂ as thealkylating agent (alkylating agent B) in solid phase (Sd; left) andsolution phase (Sn; right) methods of synthesis. These steps are similarto the steps of coupling and cyclization using alkylating agent Adescribed above. Step A involves the alkylation of the amino acidresidue (1_(BSd) or 1_(BSn)). As in FIGS. 1A-1B, PG₃, which is aprotecting group for the protection of an amine, allows the alkylatingagent to react with the hydrogen on the amine. Ns is a preferredprotecting group for this purpose. PG₃ can then be replaced withhydrogen to facilitate coupling with another residue. Typically, a mildbase is used during alkylation to facilitate hydrogen removal. Suitablebases include those described supra.

Step B in FIG. 1C involves the coupling of a second amino acid residue(2_(BSd) or 2_(BSn)) to the alkylated amino acid residue (1′_(BSd) or1′_(BSn)). In step C, the coupled amino acid residues (3_(BSd) or3_(BSn)) are cyclized upon the simultaneous or sequential addition of anacid and a hydride donor. The acid is one that removes the protectinggroup to provide for cyclization with the aldehyde. Suitable acidsinclude TFA, HCl, HBr, HCOOH, and CH₃COOH. The hydride donor ensuresthat the cyclization reaction takes place in excess hydrogen so theresulting ring is saturated. Suitable hydride donors includetriethylsilane and NaBH₃CN.

FIGS. 1E-1F and 1G-1H depict coupling and cyclization using X—(CH₂)₂—X)(alkylating agent C) or X—(CH₂)₂—OH (alkylating agent D), respectively,in solid phase (Sd; left) and solution phase (Sn; right) methods ofsynthesis. Using either agent C or D, step A involves the coupling oftwo amino acid residues (1_(CSd/CSn)+2_(CSd/CSn)→3_(CSd/CSn);1_(DSd/DSn)+2_(DSd/DSn)→3_(DSd/DSn)). Similar to the previous methods,PG₃ allows the alkylating agent to react with the hydrogen on the amineduring alkylation. Ns is preferred. PG₃ can be present as PG in compound2_(CSd) or PG₁ in compound 2_(CSn), or can be added after coupling.

Step B involves the alkylation of one of the coupled amino acid residues(3_(CSd/CSn) or 3_(DSd/DSn)). Typically, a mild base is used duringalkylation to facilitate hydrogen removal. Suitable bases includetriethylamine, N,N-diisopropylethylamine,1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 2,4,6-trimethylpyridine,potassium carbonate, and cesium carbonate. In Step C, the alkylatedcoupled amino acid residues (3′_(CSd/CSn) or 3′_(DSd/DSn)) are cyclizedupon the addition of a base. Suitable bases include triethylamine,N,N-diisopropylethylamine, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU),2,4,6-trimethylpyridine, potassium carbonate, and cesium carbonate.

The present invention may be further illustrated by reference to thefollowing examples.

EXAMPLES Example 1 Materials and Reagents

Commercial-grade reagents and solvents were used without furtherpurification except as indicated. All reactions were stirredmagnetically; moisture-sensitive reactions were performed under nitrogenin flame-dried glassware. Unless indicated, all reactions were performedat 25° C. Thin-layer chromatography (TLC), using ethyl acetate: hexane,diethyl ether: ethyl acetate, diethyl ether: hexane, DCM: methanol assolvent systems, was used to monitor reactions. Visualization wasaccomplished by either ultraviolet light or immersing the plate in 1%aqueous solution of potassium permanganate followed by heating. Flashchromatography with silica gel was performed following the conditionsdescribed by Still et al., J. Org. Chem. 43, 2923-2925 (1978), which ishereby incorporated by reference in its entirety. Solvents were removedby rotary evaporation under reduced pressure. Where appropriate, theresidue was further dried using vacuum. One-dimensional Proton (400 MHz)and carbon (100 MHz) NMR spectra were obtained on a Bruker AV-400spectrometer. Two-dimensional ¹H NMR spectra were obtained on a BrukerAV-600 (600 MHz) spectrometer. Proton chemical shifts are reported asvalues relative to tetramethylsilane (0.00 ppm) or the particularsolvent used in the experiment. Carbon chemical shifts are reported asvalues relative to the solvent used in the experiment (CDCl₃; 77.0 ppm).Data is reported as follows: chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublet,ddt=doublet of doublet of triplet, and br=broad), coupling constant, andintegration. The following abbreviations are used in the examplesdescribed infra: DCM=dichloromethane, THF=tetrahydrofuran,DIPEA=N,Ndiisopropylethylamine, TEA=triethylamine, TFA=trifluoroaceticacid, HOBt=hydroxybenzotriazole, DCC=N,N′-dicyclohexylcarbodiimide.

Example 2 Synthesis of Oligooxopiperazine 1a

A schematic of oligooxopiperazine 1a synthesis via the reductiveamination route (Tong et al., J. Org. Chem. 65:2484-2493 (2000), whichis hereby incorporated by reference in its entirety), is shown in Scheme1 below.

The synthesis of (S)—N-Allyl-Leu-OMe (5a) was carried out as follows.Allyl bromide (137.0 mmol, 11.6 mL) was added to a solution of H-Leu-OMe(55.0 mmol, 10.0 g), DIVIF (120 mL) and TEA (192.0 mmol, 26.5 mL) at 0°C. The resulting mixture was warmed to 25° C. and stirred for 48 h. Thereaction mixture was diluted with water (250 mL) and extracted withdiethyl ether (3×, 250 mL). The combined organic layers were washed withsaturated aqueous sodium bicarbonate and saturated brine, dried withMgSO₄ and concentrated under vacuum. The residue was purified by columnchromatography (15% ethyl acetate in hexane) to afford compound 5a as alight yellow oil (7.4 g, 73%). ¹H NMR (400 MHz, CDCl₃) 5.88 (ddt,J=17.1, 10.2, 6.1 Hz, 1H), 5.19 (dd, J=17.1, 1.5 Hz, 1H), 5.09 (dd,J=10.2, 1.5 Hz, 1H), 3.65 (s, 3H), 3.25 (t, J=7.4 Hz, 1H), 3.15 (ddt,J=6.2, 1.4 Hz, 1H), 2.96 (ddt, J=6.2, 1.4 Hz, 1H), 1.72 (m, 1H), 1.42(t, J=6.7 Hz, 2H), 0.88 (d, J=6.6 Hz, 3H), 0.78 (d, J=6.7 Hz, 3H); ¹³CNMR (100 MHz, CDCl₃) 176.4, 136.3, 116.4, 59.0, 51.5, 50.7, 42.8, 24.9,22.6, 22.3; HRMS m/z for C₁₀H₁₉NO₂[M+H]⁺, calcd 186.1494. found186.1486.

The synthesis of Boc-Ala-N(allyl)-Leu-OMe (2a) was carried out asfollows. A solution of Boc-Ala-OH (80.0 mmol, 15.1 g), HOBt (80.0 mmol,10.8 g) and DCC (80.0 mmol, 16.5 g) in DMF (200 mL) was stirred at 25°C. After 15 min, a solution of 5a (40.0 mmol, 7.4 g) in DMF (5 mL) wasadded, and the resulting mixture heated at reflux overnight. Thereaction mixture was cooled to 25° C., diluted with 400 mL of water andextracted with diethyl ether (250 mL, 3×). The combined organic layerswere sequentially washed with 1M NaOH (250 mL, 3×), water (250 mL, 3×),1M HCl (250 mL, 3×), and saturated brine (250 mL), dried with anhydrousMgSO₄ and concentrated under vacuum. The residue was purified by columnchromatography using 20% ethyl acetate in hexane. The purified productyielded compound 2a as a yellow oil (6.2 g, 44%). ¹H NMR (400 MHz,CDCl₃) 5.90-5.80 (m, 1H), 5.25-5.15 (m, 2H), 5.04 (q, J=5.2 Hz, 1H),4.55 (t, J=7.3 Hz, 1H), 3.94 (d, J=5.3 Hz, 2H), 3.62 (s, 3H), 1.73-1.60(m, 2H), 1.60-1.50 (m, 1H), 1.45 (s, 9H), 1.21 (d, J=6.8 Hz, 3H), 0.84(d, J=7.9 Hz, 3H), 0.78 (d, J=6.7 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)174.4, 172.2, 155.1, 134.2, 117.7, 79.6, 55.5, 52.1, 48.4, 46.7, 37.7,28.3, 24.7, 22.9, 21.8, 19.0; HRMS m/z for C₁₈H₃₂N₂O₅ [M+Na]⁺, calcd379.2209. found 379.2210.

The synthesis of (AlaLeu)Oxopiperazine methyl ester (3a) was carried outas follows. Ozone was bubbled into a solution of 2a (17.4 mmol, 6.21 g)in anhydrous methanol (200 mL) at −78° C. and ambient pressure. Thereaction mixture turned light blue after 2 h. After an additional 30min, nitrogen was bubbled into the solution until the blue colordisappeared. Dimethyl sulfide (61.0 mmol, 4.5 g) was added and themixture was stirred at room temperature. After 16 h, the mixture wasconcentrated under vacuum, and the residue (6.2 g) was dissolved in DCM(125 mL), and triethylsilane (34.6 mmol, 5.5 mL) and TFA (260.0 mmol,19.3 mL) was added. The reaction mixture was stirred for 24 h at 25° C.and then concentrated under vacuum. The residue was redissolved in DCM(60 mL) and TEA (60 mL) at 0° C. and stirred at 25° C. After one hour,the solvent was concentrated under vacuum. The residue was dissolved inDCM and the organic layer was washed with saturated aqueous sodiumbicarbonate. The aqueous layer was washed 3× with DCM. The residue waspurified by column chromatography (95% diethyl ether 5% methanol and0.1% TEA) to obtain compound 3a as a colorless oil (2.6 g, 81%). ¹H NMR(400 MHz, CDCl₃) 5.20 (t, J=8.2 Hz, 1H), 3.68 (s, 3H), 3.53 (q, J=6.8Hz, 1H), 3.28-3.20 (m, 2H), 3.10-3.02 (m, 2H), 1.63 (t, J=7.5 Hz, 2H),1.52-1.46 (septet, J=7.8 Hz, 1H), 1.32 (d, J=6.9 Hz, 3H), 0.88 (d, J=6.7Hz, 3H), 0.78 (d, J=6.5 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) 172.3, 171.4,55.4, 53.8, 52.2, 44.9, 42.2, 36.7, 24.9, 23.2, 21.3, 18.99; HRMS m/zfor C₁₂H₂₂N₂O₃[M+Na]⁺, calcd 265.1528. found 265.1523.

The synthesis of Boc-(AlaLeu)oxopiperazine-methyl ester (4a) was carriedout as follows. To a solution of 3a (6.6 mmol, 1.6 g) in DCM (22 mL) at0° C. was added 4-methylmorpholine (10.0 mmol, 1.1 mL) and ditert-butyldicarbonate (16.6 mmol, 3.6 g) in 50 mL of DCM. The mixture was allowedto warm to 25° C. and then heated at reflux. After 6 h, the mixture wasconcentrated and the residue purified by column chromatography (40%hexane in diethyl ether) to yield 2.2 g (98%) of compound 4a as acolorless oil. ¹H NMR (400 MHz, CDCl₃) 5.21 (q, J=5.72 Hz, 1H), 4.51(br, 1H), 3.89 (br, 1H), 3.64 (s, 3H), 3.46-3.35 (m, 1H), 3.26 (br, 1H),3.16-3.11 (m, 1H), 1.73-1.60 (m, 2H), 1.45 (br, 1H), 0.41 (s, 9H), 1.3(d, J=6.9 Hz, 3H), 0.88 (d, J=6.7 Hz, 3H), 0.78 (d, J=6.8 Hz, 3H); ¹³CNMR (100 MHz, CDCl₃) 172.2, 168.0, 151.3, 78.3, 52.1, 40.3, 34.2, 25.8,22.5, 20.6, 18.8, 15.5; HRMS m/z for C₁₇H₃₀N₂O₅ [M+Na]⁺, calcd 365.2052.found 365.2049.

The synthesis of oxopiperazine dimer (1a) was carried out as follows. Tosolution of 4a (6.5 mmol, 2.2 g) in THF/MeOH/H₂O (12:4:1, total volumeof 120 mL) at 0° C. was added lithium hydroxide monohydrate (16.3 mmol,0.7 g). The mixture was stirred for 2 h at 0° C. and then acidified topH 3 with saturated aqueous sodium bisulfate. The mixture wasconcentrated and the residue was dissolved in ethyl acetate and washedwith brine (2:1). The aqueous layer was extracted 3× with ethyl acetate,the combined organic layers were dried with anhydrous sodium sulfate,and concentrated under vacuum to yield 2.2 g of product. The residue wasused in the next step without further purification.

A portion of the residue from above (1.3 mmol, 0.40 g), HOBt (2.6 mmol,0.40 g) and DCC (1.3 mmol, 0.30 g) were dissolved in 50 mL of DMF. Thereaction mixture was stirred for 15 min at room temperature followed bythe addition of 3b (0.6 mmol, 0.3 g) in DMF (5 mL). The reaction mixturewas heated at 55° C. for 48 h. Then, the reaction mixture was cooled to25° C. and diluted with 100 mL of water and extracted with diethyl ether(100 mL, 3×). The combined organic layers were washed sequentially with1M NaOH (50 mL, 3×), water (50 mL), 1M HCl (50 mL, 3×), and brine (50mL). The solution was dried with anhydrous MgSO₄ and concentrated undervacuum. The residue was purified by column chromatography with 20% ethylacetate in hexane to yield compound 1a as a yellow oil (0.2 g, 73%). ¹HNMR (400 MHz, CDCl₃) 7.29-7.08 (m, 10H), 5.31 (t, J=7.2 Hz, 1H),5.25-5.15 (m, 1H), 5.03 (s, 2H), 4.65 (t, J=7.0 Hz, 1H), 4.46 (br, 1H),3.77-3.72 (m, 2H), 3.69 (s, 3H), 3.38-3.33 (m, 2H), 3.31-3.15 (m, 4.5H),3.15-2.92 (m, 3.5H), 1.50 (t, J=7.1 Hz, 2H), 1.38 (s, 9H), 1.34 (d,J=7.0 Hz, 3H), 1.34-1.15 (m, 5H), 1.02-0.93 (m, 2H), 0.87 (d, J=6.6 Hz,3H), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) 168.0, 167.2,166.1, 166.0, 153.9, 151.1, 134.2, 133.6, 126.3, 126.2, 126.0, 125.6,124.6, 113.4, 76.4, 64.1, 54.9, 53.3, 50.8, 50.0, 47.2, 41.1, 39.0,38.3, 38.2, 35.3, 34.9, 32.0, 29.2, 25.8, 22.3, 20.4, 20.2, 19.8, 15.6;HRMS m/z for C₄₂H₅₉N₅O₉ [M+Na]⁺, calcd 800.4210. found 800.4248.

Example 3 Synthesis of Oligooxopiperazine 1b

The synthesis of oligooxopiperazine dimer 1b of the present invention isillustrated in Scheme 1 above.

The synthesis of (S)—N-allyl-Phe-OMe (5b) was carried out as follows.Allyl bromide (140.0 mmol, 9.8 mL) was added to a solution of H-Phe-OMe(46.0 mmol, 10.0 g), DMF (130 mL) and TEA (164.0 mmol, 22.6 mL) at 0°C., and the reaction mixture was warmed to 25° C. After 48 h, thereaction mixture was diluted with 250 mL of water and extracted withdiethyl ether (200 mL, 3×). The combined organic layers were washed withsaturated aqueous sodium bicarbonate and saturated brine, dried withMgSO₄ and concentrated under vacuum. The residue was purified withcolumn chromatography (15% ethyl acetate in hexane) to afford compound5b as a light yellow oil 6.7 g (66%). ¹H NMR (400 MHz, CDCl₃) 7.32-7.16(m, 5H), 5.83 (ddt, J=17.1, 10.2, 6.1 Hz, 1H), 5.14 (dd, J=17.1, 1.5 Hz,1H), 5.09 (dd, J=10.2, 1.5 Hz, 1H), 3.64 (s, 3H), 3.56 (t, J=6.8 Hz,1H), 3.26 (ddt, J=6.8, 1.4 Hz, 1H), 3.15 (ddt, J=6.8, 1.4 Hz, 1H), 2.96(d, J=6.9 Hz, 2H), 1.59 (br, 1H); ¹³C NMR (100 MHz, CDCl₃) 175.0, 137.2,136.0, 129.2, 128.4, 126.8, 116.5, 62.0, 51.6, 50.6, 39.7; HRMS m/z forC₁₃H₁₇NO₂ [M+H]⁺, calcd 220.1338. found 220.1344.

The synthesis of Boc-Lys(Z)—N(allyl)-Phe-OMe (2b) was carried out asfollows. A solution of Boc-Lys(Z)—OH (29.7 mmol, 11.3 g), HOBt (29.7mmol, 4.0 g) and DCC (29.7 mmol, 6.1 g) in 200 mL of DMF was stirred at25° C. After 15 min, a solution of 5b (22.8 mmol, 5.0 g) in DMF (5 mL)was added. The mixture was heated at 55° C. After 48 h, the mixture wascooled to 25° C., diluted with 400 mL of water and extracted withdiethyl ether (300 mL, 3×). The combined organic layers weresequentially washed with 1M NaOH (400 mL, 3×), water (400 mL), 1M HCl(400 mL, 3×), and brine (400 mL). The organic layer was dried with MgSO₄and concentrated under vacuum. The residue was purified by columnchromatography using 20% ethyl acetate in hexane. The purified productyielded compound 2b as a yellow oil (3.5 g, 26%). ¹H NMR (400 MHz,CDCl₃) 7.37-7.14 (m, 10H), 5.63-5.53 (m, 1H), 5.22-5.08 (m, 4H), 5.07(s, 2H), 5.01 (br, 1H), 4.47-4.39 (m, 2H) 3.91-3.81 (br, 1H), 3.69 (s,3H), 3.50-3.31 (m, 2H), 3.21-3.09 (m, 2H), 1.77 (br, 2H), 1.57-1.48 (m,4H), 1.41 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) 170.8, 168.9, 154.5, 153.6,135.7, 134.7, 131.2, 127.6, 127.4, 126.9, 126.6, 126.3, 126.2, 125.0,116.8, 114.5, 77.8, 64.7, 58.7, 50.4, 49.4, 48.3, 38.9, 32.9, 31.3,27.4, 26.4, 20.6. HRMS m/z for C₃₂H₄₃N₃O₇ [M+Na]⁺, calcd 604.2999. found604.3005.

The synthesis of (Lys^(z)Phe)Oxopiperazine-methylester (3b) was carriedout as follows. Ozone was bubbled through a solution of 2b (3.3 mmol,1.9 g) in anhydrous methanol (12 mL) at −78° C. and ambient pressure.The reaction mixture turned light blue after 2 h. After an additional 30min, nitrogen was bubbled through until the blue color disappeared.Dimethyl sulfide (11.6 mmol, 0.9 mL) was added to the mixture and thereaction was stirred for 12 h at 25° C. The reaction mixture wasconcentrated under vacuum, and the residue (1.9 g) was redissolved in23.7 mL of DCM, and triethylsilane (6.7 mmol, 1.1 mL) and TFA (49.9mmol, 3.7 mL) were added. The mixture was stirred for 24 h at 25° C. andthen concentrated under vacuum. The residue was dissolved in DCM (12 mL)and TEA (12 mL) at 0° C. and stirred for 1 h at 25° C. The solution wasre-concentrated under vacuum and the residue dissolved in DCM (200 mL).The DCM solution was washed with saturated aqueous sodium bicarbonate(150 mL). The aqueous layer was extracted with DCM (150 mL). The organiclayers were combined, dried over anhydrous MgSO₄, and concentrated. Theresidue was purified with column chromatography (95% diethyl ether, 5%methanol and 0.1% of TEA) to obtain compound 3b as a colorless oil (0.9g, 80%). ¹H NMR (400 MHz, CDCl₃) 7.26-7.09 (m, 10H), 5.07-5.03 (m, 1H),5.00 (s, 2H), 4.84 (br, 1H), 3.64 (s, 3H), 3.45-3.26 (m, 2H), 3.26-3.21(m, 1H), 3.19-3.17 (m, 3H), 3.03-2.99 (m, 2H), 2.96-2.86 (m, 1H),1.58-1.45 (m, 3H), 1.34-1.31 (m, 2H), 1.16-0.95 (br, 2H); ¹³C NMR (100MHz, CDCl₃) 170.0, 169.5, 155.4, 135.9, 135.7, 127.8, 127.7, 127.5,127.4, 127.1, 127.0, 125.8, 65.5, 58.0, 57.2, 51.3, 45.6, 40.8, 39.6,33.1, 30.7, 28.6, 21.1; HRMS m/z for C₂₆H₃₃N₃O₅ [M+H]⁺, calcd 468.2498.found 468.2500.

The synthesis of Boc-Oxopiperazine-methylester (4b) was carried out asfollows. To a solution of 3b (3.2 mmol, 1.5 g) in 11.7 mL of DCM at 0°C. was added 4-methylmorpholine (4.8 mmol, 0.5 mL) and di-tert-butyldicarbonate (8.0 mmol, 1.8 g) in 24 mL of DCM. The mixture was allowedto warm to 25° C. and then heated at reflux for 6 h. The mixture wasconcentrated and the residue was purified by column chromatography (40%hexane in diethyl ether) to yield 1.7 g (94%) of compound 5c as acolorless oil. ¹H NMR (400 MHz, CDCl₃) 7.41-7.08 (m, 10H), 5.30-5.26 (m,1H), 5.02 (s, 2H), 4.74 (br, 1H), 4.32 (br, 1H), 3.68 (s, 3H), 3.35-3.29(m, 1H), 3.16-3.08 (m, 1H), 3.07-2.93 (m, 5H), 1.45-1.29 (m, 5H), 1.34(s, 9H), 1.13-0.93 (br, 2H); 13C NMR (100 MHz, CDCl₃) 170.7, 168.9,156.4, 153.9, 136.7, 136.4, 128.9, 128.6, 128.5, 128.1, 128.0, 127.0,80.6, 66.6, 57.2, 52.5, 43.3, 40.8, 34.4, 32.2, 28.3, 22.8; HRMS m/z forC₃₁H₄₁N₃O₇ [M+Na]⁺, calcd 590.2842. found 590.2845.

The synthesis of oxopiperazine dimer (1b) was carried out as follows.Lithium hydroxide monohydrate (4.4 mmol, 0.2 g) was added to solution of4b (1.8 mmol, 1 g) in 12:4:1 THF/MeOH/H₂O (32 mL) at 0° C. The reactionmixture was stirred for 2 h at 0° C. and then acidified to pH 3 withsaturated aqueous sodium bisulfate. The reaction mixture wasconcentrated and the residue was dissolved in ethyl acetate (15 mL) andwashed with brine (10 mL). The aqueous layer was extracted with ethylacetate (15 mL), and the combined organic layers were dried withanhydrous sodium sulfate and concentrated under vacuum to yield 1.1 g ofresidue. The residue was used in the next step without furtherpurification.

A portion of the residue from above (0.6 mmol, 0.3 g), HOBt (1.2 mmol,0.2 g) and DCC (0.6 mmol, 0.1 g) were dissolved in 100 mL of DMF. Thereaction mixture was stirred for 15 min at 25° C. and 3a (0.3 mmol, 0.1g) in DMF (5 mL) was added. The mixture was heated at 55° C. After 48 h,the solution was cooled to room temperature and diluted with 100 mL ofwater and extracted with diethyl ether (100 mL, 3×). The combinedorganic layers were washed sequentially with 1M NaOH (50 mL, 3×), water(50 mL), 1M HCl (50 mL, 3×) and brine (50 mL), dried with MgSO₄ andconcentrated under vacuum. The residue was purified by columnchromatography with 20% ethyl acetate in hexane to obtain compound 1b asa yellow oil (0.1 g, 70%). ¹H NMR (400 MHz, CDCl₃) 7.29-7.11 (m, 10H),5.72-5.63 (m, 1H), 5.22-5.16 (m, 1H), 5.02 (s, 2H), 4.88-4.80 (m, 1H),4.34 (br, 1H), 3.91 (br, 1H), 3.64 (s, 3H), 3.59-3.54 (m, 2H), 3.46-3.41(m, 1H), 3.38-3.21 (m, 2H), 3.15-2.90 (m, 6H), 1.93-1.57 (m, 5H),1.36-1.22 (m, 15H), 1.19-1.06 (m, 2H), 0.87 (d, J=6.6 Hz, 3H), 0.81 (d,J=6.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) 170.6, 168.5, 168.2, 167.5,166.2, 135.2, 134.9, 128.4, 128.3, 128.2, 127.6, 127.5, 127.48, 127.46,127.1, 126.0, 79.9, 65.6, 52.4, 51.4, 51.3, 51.2, 41.3, 39.8, 39.4,35.9, 35.8, 34.1, 33.9, 28.9, 28.7, 27.3, 27.27, 24.0, 22.2, 22.1, 21.9,20.2; HRMS m/z for C₄₂H₅₉N₅O₉ [M+Na]⁺, calcd 800.4210. found 800.4233.

Example 4 Synthesis of Oligooxopiperazine 1c

The synthesis of oligooxopiperazine dimer 1c of the present invention isillustrated in Scheme 1 above.

The synthesis of Boc-Leu-N(allyl)-Leu-OMe (2c) was carried out asfollows. A solution of Boc-Leu-OH (78.6 mmol, 24.1 g), HOBt (78.6 mmol,10.6 g) and DCC (78.6 mmol, 16.2 g) in 200 mL of DMF was stirred at 25°C. After 15 min, a solution of 5a (39.3 mmol, 7.3 g) was added in DMF (5mL), and the resulting mixture was heated at 55° C. for 12 h. Themixture was cooled to 25° C., diluted with 400 mL of water and extractedwith diethyl ether (300 mL, 3×). The combined organic layers were washedsequentially with 1M NaOH (500 mL, 3×), water (500 mL), 1M HCl (500 mL,3×), and brine (500 mL). The organic layer was dried with anhydrousMgSO₄ and concentrated under vacuum. The residue was purified by columnchromatography (20% ethyl acetate in hexane) to afford 2c as a yellowoil (6.2 g, 40%). ¹H NMR (400 MHz, CDCl₃) 5.92-5.74 (m, 1H), 5.22-5.16(m, 2H), 5.06-4.97 (m, 2H), 4.57-4.54 (m, 1H), 4.01-3.91 (br, 2H), 3.65(s, 3H), 1.79-1.56 (m, 3H), 1.54-1.43 (m, 3H), 1.35 (s, 9H), 0.93-0.74(m, 12H); ¹³C NMR (100 MHz, CDCl₃) 174.4, 172.2, 155.5, 134.2, 117.7,79.4, 55.4, 52.1, 49.3, 48.3, 42.4, 37.8, 28.3, 24.6, 23.5, 22.9, 21.7;HRMS m/z for C₂₁H₃₈N₂O₅ [M+H]⁺, calcd 398.2859. found 399.2862.

The synthesis of (LeuLeu)Oxopiperazine methyl ester (3c) was carried outas follows. Ozone was bubbled through a solution of 2c (11.0 mmol, 4.4g) in anhydrous methanol (75 mL) at −78° C. and ambient pressure. Thereaction mixture turned light blue after 2 h. After an additional 30min, nitrogen was bubbled through until the blue color disappeared.Dimethyl sulfide (38.5 mmol, 2.8 mL) was added and the mixture stirredfor 12 h at 25° C. The mixture was concentrated under vacuum, and theresidue was redissolved in DCM (78 mL), triethylsilane (21.9 mmol, 3.5mL) and TFA (165.0 mmol, 12.2 mL). The reaction mixture was stirred for24 h at 25° C. and then concentrated under vacuum. The residue wasdissolved in 39 mL of DCM and 39 mL of TEA at 0° C. and stirred for h at25° C. The solvent was then concentrated under vacuum. The residue wasredissolved in DCM (300 mL), and the solution washed with saturatedaqueous sodium bicarbonate (300 mL). The aqueous layer was extractedwith DCM (200 mL, ×3). The combined organic layers were concentrated andthe residue was purified with column chromatography (95% diethyl ether,5% methanol, and 0.1% of LEA) to obtain compound 3c as a colorless oil(2.2 g, 85%). ¹H NMR (400 MHz, CDCl₃) 5.26-5.22 (t, J=7.8 Hz, 1H), 3.65(s, 3H), 3.48-3.38 (m, 1H), 3.27-3.17 (m, 2H), 3.14-3.09 (m, 1H),3.02-2.95 (m, 1H), 1.83-1.76 (m, 1H), 1.74-1.61 (m, 4H), 1.52-1.41 (m,2H), 0.89-0.85 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) 172.3, 171.5, 57.7,53.8, 52.1, 44.7, 42.0, 41.6, 36.7, 24.9, 24.5, 23.5, 23.2, 21.3, 21.1;HRMS m/z for C₁₅H₂₈N₂O₃ [M+H]⁺, calcd 285.2178. found 285.2182.

The synthesis of Boc-(LeuLeu)oxopiperazine-methyl ester (4c) was carriedout as follows. To a solution of 3c (7.7 mmol, 2.2 g) in 25 mL of DCM at0° C. was added 4-methylmorpholine (11.5 mmol, 1.3 mL) and ditert-butyldicarbonate (19.2 mmol, 4.2 g) in 50 mL of DCM. The mixture was allowedto warm to 25° C., and then heated at reflux. After 6 h, the mixture wasconcentrated and the residue was purified by column chromatography (40%hexane in diethyl ether) to yield 2.9 g (97%) of compound 4c as acolorless oil. ¹H NMR (400 MHz, CDCl₃) 5.21-5.17 (m, 1H), 4.53 (br, 1H),3.95 (br, 1H), 3.66 (s, 3H), 3.44-3.34 (m, 1H), 3.23 (br, 1H), 3.15-3.10(m, 1H), 1.70-1.62 (m, 3H), 1.60-1.52 (m, 3H), 1.45 (s, 9H), 0.91-0.81(m, 12H); ¹³C NMR (100 MHz, CDCl₃) 171.6, 169.7, 154.1, 80.7, 56.2,53.6, 52.1, 41.9, 41.8, 37.7, 36.8, 28.3, 24.9, 24.6, 23.2, 22.8, 22.3,21.2; HRMS m/z for C₂₀H₃₆N₂O₅[M+Na]⁺, calcd 407.2522. found 407.2510.

The synthesis of oxopiperazine dimer (1c) was carried out as follows. Tosolution of 4c (5.4 mmol, 1.9 g) in 12:4:1 THF/MeOH/H₂O (100 mL) at 0°C. was added lithium hydroxide monohydrate (16.4 mmol, 0.7 g). Thereaction mixture was stirred for 2 h at 0° C. and then acidified to pH 3with saturated aqueous sodium bisulfate. The reaction mixture wasconcentrated and the residue was dissolved in ethyl acetate (100 mL) andwashed with brine (50 mL). The aqueous layer was extracted with ethylacetate (100 mL), and the combined organic layers were dried withanhydrous sodium sulfate and concentrated to yield 2 g of productresidue. The residue was used in the next step without furtherpurification.

A portion of the residue from above (1.8 mmol, 0.70 g), HOBt (3.5 mmol,0.50 g) and DCC (1.8 mmol, 0.40 g) were dissolved in 50 mL of DMF. Thesolution was stirred for 15 min at 25° C., and 3c (0.9 mmol, 0.3 g) wasadded in DMF (5 mL). The mixture was heated at 55° C. for 48 h and thencooled to 25° C. and diluted with water (100 mL) and extracted withdiethyl ether (100 mL, 3×). The combined organic layers were washedsequentially with 1M NaOH (50 mL, 3×), water (50 mL), 1M HCl (50 mL, 3×)and brine (50 mL), dried with MgSO₄, and concentrated under vacuum. Theresidue was purified by column chromatography with 20% ethyl acetate inhexane to obtain compound 1c as a yellow oil (0.2 g, 71%). ¹H NMR (400MHz, CDCl₃) 5.60-5.58 (t, J=7.4 Hz, 1H), 5.28-5.25 (m, 1H), 5.12-5.08(m, 1H), 4.58 (br, 1H), 4.23 (br, 1H), 4.12 (br, 1H), 3.66 (s, 3H),3.48-3.42 (m, 1H), 3.39-3.24 (m, 4H), 3.20-3.11 (m, 1H), 1.73-1.49 (m,10H), 1.45 (s, 9H), 1.44-1.36 (m, 2H), 0.97-0.88 (m, 24H); ¹³C NMR (100MHz, CDCl3) 171.8, 171.7, 169.6, 169.2, 154.1, 54.9, 54.5, 53.5, 53.3,52.3, 52.2, 42.5, 42.2, 41.7, 41.5, 41.4, 41.2, 40.6, 39.5, 37.6, 37.4,37.0, 36.8, 28.3, 28.0, 25.1, 25.0, 24.9, 24.8, 24.7; HRMS m/z forC₃₄H₆₀N₄O₇ [M+Na]+, calcd 659.4360. found 659.4350.

Example 5 Two-Dimensional NMR Spectroscopy of Oligooxopiperazine 1a

COSY spectrum of 1a was recorded on a Bruker Avance 400 at 20° C. bycollecting 2048 complex data points in the t₂ domain by averaging 32scans and 256 increments in the t₁ domain with States-TPPI mode. Theoriginal free induction decays (FIDs) were zero-filled to give a finalmatrix of 1024 by 1024 real data points. A 0° sine-bell window functionwas applied in both dimensions. NOESY spectrum of 1a was recorded on aBruker Avance 600 at 20° C. by collecting 4096 complex data points inthe t₂ domain by averaging 48 scans and 512 increments in the t₁ domainwith States-TPPI mode and the mixing time of 750 ms. The original freeinduction decays (FIDs) were zero-filled to give a final matrix of 2048by 1024 real data points. A 90° sine-square window function was appliedin both dimensions. All the data were processed and analyzed usingBruker TOPSPIN 1.3 program.

Example 6 Circular Dichroism (CD) Spectroscopy Studies

CD spectra were recorded on AVIV 202SF CD spectrometer equipped with atemperature controller using 1 mm length cells and a scan speed of 15nm/min. The spectra were averaged over 10 scans with the baselinesubtracted from analogous conditions as that for the samples. Thesamples were prepared in acetonitrile or methanol with the final peptideconcentration of 100 M. The amount of oxopiperazines were determined bydry weight.

Example 7 Conformational Analysis of Oligooxopiperazines

The present invention relates to the design and synthesis of nonaromatichelix mimetics which feature a chiral backbone and are easilysynthesized from α-amino acids. The piperazine skeleton was anattractive design choice because it is considered a privileged scaffoldfor peptidomimetic research and drug discovery (Patchett et al., Ann.Rep. Med. Chem. 35: 289-298 (2000), which is hereby incorporated byreference in its entirety). Specifically, the 2-oxopiperazine and thediketopiperazines have a rich history in medicinal chemistry and areconsidered to be “drug-like” scaffolds (Herrero et al., J. Org. Chem.67:3866-3873 (2002); Kitamura et al., J. Med. Chem. 44:2438-2450 (2001);Gante, J “Peptidomimetics—Tailored Enzyme-Inhibitors,” Angew. Chem. Int.Ed. Engl. 33:1699-1720 (1994); Giannis et al., Angew. Chem. Int. Ed.32:1244-1267 (1993), which are hereby incorporated by reference in theirentirety). Initial computational studies of the oligooxopiperazinespredicted stable structures due to the conformational constraintsinherent in the system. Molecular modeling studies indicate that anoxopiperazine dimer spans the length of an 8mer α-helix and superimposesamino acid functionality onto the i, i+4, and i+7 residues of the helix(FIG. 2B). Oligooxopiperazines do not contain hydrogen bond donors inthe backbone; however, this omission is not expected to be detrimentalfor helix mimetics because helices typically do not utilize backbonehydrogen bonding functionality for interaction with other biomolecules.

The analysis of oligooxopiperazines was started by searching theCambridge Structural Database for examples of oxopiperazine derivatives.This search resulted in five hits (CSD codes: KEMXUV, ZOZTUD, ZARZOH,FOBFEH, and KEMXUV) of single piperazine ring systems relevant to thesystem. Although this is a narrow set to base hypotheses upon, thesehits provided invaluable insights regarding the yo and yr dihedralangles favored in the amino acid residue linking two piperazine ringsand corroborated the molecular modeling calculations (FIGS. 3A-3C).

The oxopiperazine rings may adopt the half-chair or the boatconformation, but the half-chair conformation is substantially lower inenergy, ˜2.9 Kcal/mol (FIG. 3B). A dimer of oxopiperazines containsthree rotatable bonds φ, ψ, and ω. The tertiary amide bond may adopt acis or trans amide conformation like proline as shown in FIG. 3C.Macromodel calculations suggest that the trans conformation is roughly 1Kcal/mol more stable than the cis conformation in tetraalanine systems.The trans to cis ratio is expected to increase in dimers built frombulkier amino acid residues.

To examine the preferred φ and ψ dihedral angles in an oxopiperazinedimer, dimer 30 (see FIG. 7), the “dihedral drive” functionality inMacromodel was utilized (Mohamadi et al., J. Comp. Chem. 11:440-467(1990), which is hereby incorporated by reference in its entirety). Theresults of these calculations intimate a limited number and a narrowrange of φ and ψ values in the lowest energy conformations (Table 1below and FIGS. 7A-7B). Importantly, the dihedral angles predicted byMacromodel were also found in the crystal structures of relevantcompounds in CSD (Table 1). The calculations indicate thatoligooxopiperazines will favor φ and ψ angles of −128°±25° and 76°±15°,respectively. The favored y values show direct correlation with allyl1,2 and 1,3 strains.

TABLE 1 Calculated Low Energy and Values for Oxopiperazine Dimer 30. 30

Dihedral Relative energy Cambridge structure angle (°) (Kcal/mol)database code* φ −150 0.95 — −128 0 KEMXUV, ZOZTUD −90 1.26 ZARZOH ψ 600.64 — 76.76 0 ZARZOH 90 0.34 — 120 1.87 — *the corresponding dihedralvalue was found in the indicated CSD structure.

The predicted low energy structure of the oxopiperazine dimer arraysfunctionality to match side chain patterns on a canonical α-helix (FIG.2). Similarly, the predicted low energy structure of the oxopiperazinetrimer arrays functionality to match side chain patterns on a canonicalα-helix (FIG. 8). Positions 1, 2, 3, and 4 overlay well onto the i+1,i+2, i+3 and i+7 residues on a 10mer α-helix; while the i+1, i+4 and i+7positions are best mimicked by positions 1,2, and 5 of anoligooxopiperazine (FIG. 8). This level of structural versatility hasnot been observed with other nonpeptidic helix mimetics, which typicallyonly mimic one face of the helix (Davis et al., Chem. Soc. Rev.36:326-334 (2007); Yin et al., Angew. Chem. Int. Ed., 44: 4130-4163(2005), which are hereby incorporated by reference in their entirety.

Oligooxopiperazines 1a-c were designed to test the impact of differentside chain combinations on the stability of the oxopiperazine dimerconformation. Several synthetic routes to piperazines are known, whichwere anticipated to allow rapid synthesis and evaluation of the desiredcompounds (Franceschini et al., Org. Biomol. Chem. 3:787-793 (2005);Tong et al., J. Org. Chem. 65:2484-2493 (2000); Sugihara et al., J. Med.Chem. 41:489-502 (1998), which are hereby incorporated by reference intheir entirety). While a number of these synthetic routes wereevaluated, it was discovered that the reductive amination routedescribed by Tong et al., J. Org. Chem., 65:2484-93 (2000), which ishereby incorporated by reference in its entirety, can afford shortoligomers in respectable yields (FIG. 4B and Scheme 1).

The solution conformation of dimers 1a-c was investigated by CDspectroscopy in methanol and acetonitrile solutions. FIGS. 5A and 5Cshow CD spectra of oxopiperazine 1a, 1b, and 1c in acetonitrile andmethanol, respectively. The CD spectra of 1a-c display double minimanear 220 and 230 nm and maxima at 200 nm. Surprisingly, the overallshape is reminiscent of CD spectra of α-helices; although, the maximaand minima are red-shifted by 10 nm. Although CD spectra of artificialsystems are often difficult to interpret (Driver et al., Org. Lett.11:3092-3095 (2009), which is hereby incorporated by reference in itsentirety), the spectra of 1a-c indicate a high degree ofpreorganization. The thermal stabilities of 1a-c were investigated bymonitoring the temperature-dependent change in the intensity of the 220nm bands in the CD spectra (FIG. 5B). A gradual increase in the signalintensity was observed at 220 nm with temperature, but the dimers retainover 70% of their room-temperature elipticity at 75° C. Similarnon-cooperative denaturation behavior has been observed with otherconformationally defined oligomers (Saludes et al., Am. Chem. Soc.131:5495-5505 (2009); Wang et al., Org. Biomol. Chem. 4:4074-4081(2006). Overall, the CD studies demonstrate that helix mimetics 1a-cadopt stable conformations confirming the molecular modeling analysis.

Two-dimensional NMR spectroscopy was also utilized to analyze theconformations adopted by 1a as a model oxopiperazine helix mimetic,specifically to determine the geometry adopted by the tertiary amidebond linking two piperazine rings. A combination of COSY and NOESYspectroscopy was used to assign ¹H NMR resonances for 1a. The NOESYspectrum reveals several NOEs in the two-ring system, which would beexpected from a trans-amide geometry in 1a but not from the cis-amideconformation (FIG. 6A). NOE crosspeaks were not observed between protonson neighboring piperazine rings (FIG. 6B). This absence of NOEs isexpected based on the proposed low energy conformation in which theseprotons lie outside the 5 Å distance typically required to observe thenuclear Overhouser effect. Thus, the NOESY studies strongly corroboratethe modeling analysis. Significantly, the NMR spectra did not displaypeaks indicative of a minor cis-amide isomer, suggesting that the transconformation is substantially more stable than the cis analog.

Example 8 Representative Solid Phase Synthesis of Oligooxopiperazines

An alternative route of oligooxopiperazine synthesis was investigated.Scheme 2 below illustrates a representative solid phase synthesis schemefor the synthesis of oligooxopiperazine dimers (i.e., dimers A, B, andC) and trimers. FIGS. 9A-9D show the predicted structures of theoligooxopiperazine dimers A, B, and C, and trimer as they overlay withthe target α-helix. Exemplary dimers and trimers produced via thissynthesis approach are shown in Tables 2 and 3 below. The biologicalprotein target of the oligooxopiperazine, the helical sequence of thetarget protein, and the oligooxopiperazine structure are provided inTables 2 and 3.

TABLE 2 Exemplary Oligooxopiperazines and their Helical Targets Sequenceof Helical Target Model Type Partner Oligooxopiperazine Structure HDM2Trimer p53₁₇ ₋ ₂₈ ET F SDL W KL L PE (SEQ ID NO: 186)

HDM2 Dimer A p53₁₇₋₂₈ ET F SDL W KL L PE (SEQ ID NO: 186)

p300-TAZ1 Dimer B Hifl₁₄₀₋₁₄₇ E L LRA L D O (SEQ ID NO: 187)

p300-KIX Dimer C cMyb₉₁₋₁₀₃ RIKE L EL LL MS T E (SEQ ID NO: 188)

p300-SID Dimer C p16₀₅₋₁₆ D ERA LL DQ L HTL (SEQ ID NO: 189)

p300-IBid Dimer C IRF3₃₇₂₋₃₈₁ LRA L VE M AR V (SEQ ID NO: 190)

X = H, COCH₃, amino acid; Y = OH, NH₂, OMe, amino acid bold residuesindicate key residues for binding.

TABLE 3 Exemplary Oligooxopiperazines and their Helical Targets WildType Sequence Target Model Type Helical Domain* OligooxopiperazineStructure* p53/MDM2 Trimer p53₁₇ ₋ ₂₈ ET F SDL W KL L PE (SEQ ID NO:186)

p53/MDM2 Dimer A p53₁₇₋₁₈ ET F SDL W KL L PE (SEQ ID NO: 186)

Hifl/p300 Dimer B Hifl₁₄₀₋₁₄₇ E L LRA L D O (SEQ ID NO: 187)

cMyb/KIX Dimer C cMyb₉₁₋₁₀₃ RIKE L EL LL MS T E (SEQ ID NO: 188)

*bold residues are critical for binding of the helix to the proteinpartner

Example 9 Biological Potential of Oligooxopiperazines

The potential of the oligooxopiperazine molecules of the presentinvention to inhibit protein—protein interactions in which helices playkey roles at the interfaces will be tested using the Bcl-xL/Bak-BH3(Sattler et al., Science 275:983-986 (1997), which is herebyincorporated by reference in its entirety) and p53/Mdm2 (Kussie et al.,Science 274:948-953 (1996), which is hereby incorporated by reference inits entirety) complexes as targets. Both of these complexes areintimately involved in regulating the crucial process of programmed celldeath. These complexes have been chosen for the initial foray into thecontrol of protein—protein interactions with oligooxopiperazines becausethese protein complexes have been targeted with several differentstrategies, including small molecules, allowing the evaluation of thesuitability of this approach (Murray et al., Biopolymers 88:657-686(2007); Ernst et al., Angew. Chem. Int. Ed. Engl. 42: 535-539 (2003);Walensky et al., Science 305:1466-70 (2004); Gemperli et al., J. Am.Chem. Soc. 127:1596-7 (2005); Sadowsky et al., J. Am. Chem. Soc. 129:139-154 (2007); Davis et al., Chem. Soc. Rev. 36:326-334 (2007), whichare hereby incorporated by reference in their entirety).

Oligooxopiperazine 38 (FIG. 10C) has been designed and synthesized tomimic the p53 helix. This helix features three hydrophobic residuesphenylalanine, tryptophan, and leucine on the same face (at positions i,i+4, and i+7) and it binds in a deep hydrophobic cleft of Mdm2 (FIG.10A). Modeling studies suggest that oligooxopiperazine trimer positions1,2, and 5, respectively, would overlay well onto i, i+4, and i+7positions of an α-helix (FIG. 10B). Accordingly, oligooxopiperazine 38was designed to display phenylalanine, tryptophan and leucine sidechains at position 1, 2, and 5 of the trimer, respectively (FIG. 10C).For these preliminary studies, oligooxopiperazine trimer 39, which lacksthe key tryptophan residue at position 2, has also been synthesized.This negative control will allow assessment of the specificity ofoligooxopiperazines for their targets. A oxopiperazine trimer has thepotential to display six residues and mimic a 10-mer helix. In thisfirst generation study only three key residues from p53 will be importedinto the oligoxopiperazine scaffold; in subsequent studies the otherresidues from the p53 sequence will also be introduced and studied in aniterative manner.

In summary, through rational design and synthesis, a new class ofnonpeptidic α-helix mimetics have been developed. NMR and circulardichroism spectroscopies provide compelling evidence thatoligooxopiperazine dimers adopt stable conformations that reproduce thearrangement of i, i+4, and i+7 residues on an α-helix. Given theimportance of the helix conformation in protein—protein interactions,and the potential of nonpeptidic scaffolds that mimic this conformation,these oxopiperazine scaffolds will offer attractive new tools forchemical biology (Jochim and Arora, Mol. BioSyst. 5:924-926 (2009);Jones and Thornton, Proc. Natl. Acad. Sci U.S.A. 93:13-20(1996), whichare hereby incorporated by reference in their entirety). Oxopiperazinehelix mimetics have the potential to disrupt chosen protein—proteininteractions.

1. An oligooxopiperazine of Formula I:

wherein: each of R₁, R₂, R₃, and R₄ is independently an amino acid sidechain, H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R isindependently H, an alkyl, or an aryl; each R₆ is independently H,N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R isindependently H, an alkyl, or an aryl; A is X₁, wherein: X₁ is H, COR′,CO₂R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a protecting group for protection of an amine, a targetingmoiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; and B is Y or D,wherein: Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a protecting group for protection of acarboxylic acid, a targeting moiety, or a tag; wherein: R′ is H, analkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targetingmoiety, or a tag; and each R′″ is independently H, CO₂R′, CONHR′, analkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targetingmoiety, or a tag; and D is a moiety of the formula

 wherein: R₅ is an amino acid side chain, H, N(R)₂, OR, halogen, analkyl, or an aryl; wherein each R is independently H, an alkyl, or anaryl; R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each Ris independently H, an alkyl, or an aryl; and E is X₂ or F, wherein: X₂is H, COR′, CO₂R′, CONHR′, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a protecting group for protection of an amine,a targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; andF is a moiety of the formula

 wherein:  R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; whereineach R is independently H, an alkyl, or an aryl;  R₇ is an amino acidside chain; and  Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protectionof a carboxylic acid, a targeting moiety, or a tag; wherein:  R′ is H,an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targetingmoiety, or a tag; and  each R′″ is independently H, CO₂R′, CONHR′, analkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targetingmoiety, or a tag.
 2. The oligooxopiperazine according to claim 1,wherein the oligooxopiperazine is: (i) an oligooxopiperazine of FormulaIA:

 or (ii) an oligooxopiperazine of Formula IB:

3-7. (canceled)
 8. The oligooxopiperazine according to claim 1, whereinthe oligooxopiperazine mimics an α-helix involved in a protein—proteininteraction.
 9. The oligooxopiperazine according to claim 8, wherein theoligooxopiperazine is: (i) an oligooxopiperazine of Formula IA:

 or (ii) an oligooxopiperazine of Formula IB:


10. The oligooxopiperazine according to claim 9, wherein: (i) theoligooxopiperazine is an oligooxopiperazine of Formula IA and R₁, R₂,R₄, and R₅ mimic the amino acid side chain of, respectively, residues i,i+4, i+6, and i+7 of the α-helix; (ii) the oligooxopiperazine is anoligooxopiperazine of Formula IB and R₁, R₂, and R₄ mimic the amino acidside chain of, respectively, residues i, i+4, and i+7 of the α-helix; or(iii) the oligooxopiperazine is an oligooxopiperazine of Formula IB andR₁, R₂ and R₄ mimic the amino acid side chain of, respectively, residuesi, i+4, and i+6 of the α-helix. 11-19. (canceled)
 20. Theoligooxopiperazine according to claim 8, wherein the α-helix is selectedfrom the group consisting of SEQ ID NOs:1-181.
 21. A pharmaceuticalformulation comprising: an oligooxopiperazine according to claim 1 and apharmaceutically acceptable vehicle. 22-72. (canceled)
 73. Anoligooxopiperazine of Formula I:

wherein: each of R₁, R₂, R₃, and R₄ is independently an amino acid sidechain, H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R isindependently H, an alkyl, or an aryl; each R₆ is independently H,N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R isindependently H, an alkyl, or an aryl; A is X₁ or C, wherein: X₁ is H,COR′, CO₂R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a protecting group for protection of an amine, a targetingmoiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; and C is amoiety of the formula

 wherein: each X′ is independently H, COR′, CO₂R′, CONHR′, N(R″)₂, analkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targetingmoiety, or a tag; wherein: R′ is H, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; and each R″ isindependently H, CO₂R′, CONHR′, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; R₀ is an aminoacid side chain, H, N(R)₂, OR, halogen, an alkyl, or an aryl; whereineach R is independently H, an alkyl, or an aryl; and R₆ is H, N(R)₂, OR,halogen, an alkyl, or an aryl; wherein each R is independently H, analkyl, or an aryl; and B is Y wherein: Y is OR′, COR′, N(R′″)₂, analkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a protectinggroup for protection of a carboxylic acid, a targeting moiety, or a tag;wherein: R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a targeting moiety, or a tag; and each R′″ is independentlyH, CO₂R′, CONHR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a targeting moiety, or a tag.
 74. The oligooxopiperazineaccording to claim 73, wherein the oligooxopiperazine is: (i) anoligooxopiperazine of Formula IB:

 or (ii) an oligooxopiperazine of Formula IC:


75. The oligooxopiperazine according to claim 73, wherein theoligooxopiperazine mimics an α-helix involved in a protein—proteininteraction.
 76. the oligooxopiperazine according to claim 75, whereinthe oligooxopiperazine is: (i) an oligooxopiperazine of Formula IB:

 or (ii) an oligooxopiperazine of Formula IC:


77. The oligooxopiperazine according to claim 76, wherein: (i) theoligooxopiperazine is an oligooxopiperazine of Formula IB and R₁, R₂,and R₄ mimic the amino acid side chain of, respectively, residues i,i+4, and i+7 of the α-helix; (ii) the oligooxopiperazine is anoligooxopiperazine of Formula IB and R₁, R₂, and R₄ mimic the amino acidside chain of, respectively, residues i, i+4, and i+6 of the α-helix; or(iii) the oligooxopiperazine is an oligooxopiperazine of Formula IC andR₀, R₁, R₂, R₃, and R₄ mimic the amino acid side chain of, respectively,residues i, i+2, i+3, i+4, and i+7 of the α-helix.
 78. Theoligooxopiperazine according to claim 75, wherein the α-helix isselected from a group consisting of SEQ ID NOs:1-181.
 79. Apharmaceutical formulation comprising: an oligooxopiperazine accordingto claim 73 and a pharmaceutically acceptable vehicle.
 80. Anoligooxopiperazine of Formula I:

wherein: each of R₁, R₂, R₃, and R₄ is independently an amino acid sidechain, H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R isindependently H, an alkyl, or an aryl; each R₆ is independently H,N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each R isindependently H, an alkyl, or an aryl; A is X₁ or C, wherein: X₁ is H,COR′, CO₂R′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a protecting group for protection of an amine, a targetingmoiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; and C is amoiety of the formula

 wherein: each X′ is independently H, COR′, CO₂R′, N(R″)₂, an alkyl, anaryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or atag; wherein: R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a targeting moiety, or a tag; and each R″ is independentlyH, CO₂R′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, atargeting moiety, or a tag; R₀ is an amino acid side chain, H, N(R)₂,OR, halogen, an alkyl, or an aryl; wherein each R is independently H, analkyl, or an aryl; and R₆ is H, N(R)₂, OR, halogen, an alkyl, or anaryl; wherein each R is independently H, an alkyl, or an aryl; and B isY or D, wherein: Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protectionof a carboxylic acid, a targeting moiety, or a tag; wherein: R′ is H, analkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targetingmoiety, or a tag; and each R′″ is independently H, CO₂R′, an alkyl, anaryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or atag; and D is a moiety of the formula

 wherein: R₅ is an amino acid side chain, H, N(R)₂, OR, halogen, analkyl, or an aryl; wherein each R is independently H, an alkyl, or anaryl; R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; wherein each Ris independently H, an alkyl, or an aryl; and E is X₂ or F, wherein: X₂is H, COR′, CO₂R′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a protecting group for protection of an amine, a targetingmoiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; and F is amoiety of the formula

 wherein:  R₆ is H, N(R)₂, OR, halogen, an alkyl, or an aryl; whereineach R is independently H, an alkyl, or an aryl;  R₇ is an amino acidside chain; and  Y is OR′, COR′, N(R′″)₂, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a protecting group for protectionof a carboxylic acid, a targeting moiety, or a tag; wherein:  R′ is H,an alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targetingmoiety, or a tag; and  each R′″ is independently H, CO₂R′, an alkyl, anaryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or atag; with the proviso that A and B are not both, respectively, C and D.81. The oligooxopiperazine according to claim 80, wherein theoligooxopiperazine is an oligooxopiperazine of Formula IA:


82. The oligooxopiperazine according to claim 80, wherein theoligooxopiperazine is an oligooxopiperazine of Formula IB:


83. The oligooxopiperazine according to claim 80, wherein theoligooxopiperazine is an oligooxopiperazine of Formula IC:


84. The oligooxopiperazine according to claim 80, wherein theoligooxopiperazine mimics an α-helix involved in a protein—proteininteraction.
 85. The oligooxopiperazine according to claim 84, whereinthe oligooxopiperazine is an oligooxopiperazine of Formula IA:


86. The oligooxopiperazine according to claim 85, wherein R₁, R₂, R₄,and R₅ mimic the amino acid side chain of, respectively, residues i,i+4, i+6, and i+7 of the α-helix.
 87. The oligooxopiperazine accordingto claim 84, wherein the oligooxopiperazine is an oligooxopiperazine ofFormula IB:


88. The oligooxopiperazine according to claim 87, wherein R₁, R₂, and R₄mimic the amino acid side chain of, respectively, residues i, i+4, andi+7 of the α-helix.
 89. The oligooxopiperazine according to claim 87,wherein R₁, R₂, and R₄ mimic the amino acid side chain of, respectively,residues i, i+4, and i+6 of the α-helix.
 90. The oligooxopiperazineaccording to claim 84, wherein the oligooxopiperazine is anoligooxopiperazine of Formula IC:


91. The oligooxopiperazine according to claim 90, wherein R₀, R₁, R₂,R₃, and R₄ mimic the amino acid side chain of, respectively, residues i,i+2, i+3, i+4, and i+7 of the α-helix.
 92. The oligooxopiperazineaccording to claim 84, wherein the α-helix is selected from a groupconsisting of SEQ ID NOs:1-181.
 93. The oligooxopiperazine according toclaim 80, wherein the oligooxopiperazine is

wherein X is H and Y is NH₂.
 94. A pharmaceutical formulationcomprising: an oligooxopiperazine according to claim 80 and apharmaceutically acceptable vehicle.